RNA SURVEILLANCE: molecular approaches in the control of the transcripts and its

Production of mature mRNAs that encode functional proteins consists of highly complex pathways of synthesis, processing, and surveillance. Along the maturation process, the mRNA transcript is scrutinized by quality control machinery at numerous steps. The extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possible deleterious proteins. Recent advances in the understanding the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and a variety of human diseases are caused by the disturbance in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss of function and gain of function are relevant and a considerable number of different diseases exemplify the importance of the mechanistic function of the RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, which highlight the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of the therapeutic strategies. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F Introduction Messenger RNA (mRNA) mediates the transfer of the genetic information from the cell nucleus to the cytoplasm (1). The development of a mature mRNA molecule requires several multiple steps that proceed through a progressive sequence of interdependent processing events: transcription, capping, splicing, edition, 3’-end processing, and numerous events dependent on RNA-binding proteins that bind to the transcript forming an mRNA ribonucleoprotein (mRNP) complex (2). The perfect synchrony of those events is maintained by cellular quality control machinery that assures tight regulation of gene expression and consequently a healthy organism. Since the molecular aspects in the mRNA metabolism have been extensively investigated many points concerning this metabolic pathway are known. After processing, the surveillance cellular machinery try to identify prone errors in the transcripts (3,4). These errors can lead to the mRNA body degradation, avoiding disastrous consequences to the organism since the defective transcripts have the potential to be translated into aberrant and deleterious proteins. This quality control machinery serves a critical role in the maintenance of a healthy life and studies have link error-induced mRNA metabolism to several human diseases (5,6,7). Notable examples of such failures in the mRNA quality control mechanisms can be exemplified by β thalassemia (8) and Marfan syndrome (9). Certain kinds of cancers and heritable genetic diseases may also be associated with mistakes in the mRNA edition and/ or mRNA processing (5). For instance, a variety of clinical diseases are due to inappropriate 3’-end processing of the transcripts (10,11,12). Several laboratories are conducting research in RNA field in order to understand all the molecular aspects of the mRNA processing and surveillance. This research will lead to Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F the development of a more sophisticated clinical diagnosis and therapeutic approaches. This review will discuss key mechanistic features of mRNA processing and surveillance, followed by a study on the clinical perspective of those mechanisms, which illustrate how diseases can be caused by errors/ mutations of important RNA sequence elements and/ or by the pathological expression of proteins whose correspondent mRNA contains serious mistakes. Also, a brief overview of the influence of circadian rhythms on RNA processing and surveillance will be presented. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F The mRNA processing and surveillance Cotranscriptional mRNA processing: the integrated events that control gene expression In eukaryotes, including humans, DNA is transcribed initially as a precursor mRNA in the nucleus by RNA polymerase II (RNAP II) (13). Soon after RNAP II initiates the transcription, the nascent molecule is modified by the addition of a 7-methyl guanosine cap structure at the 5’-end of the RNA. The introns are also cotranscriptionally removed from the precursor mRNA; the body of this molecule has its sequence edited and the cleavage and the polyadenylation of the 3’-end also take place (2). In the course of all the processing events, a variety of mRNA-binding proteins and other factors interact with the maturing transcript (14,15), assuming a critical function in the mRNA surveillance context. Understanding of the cotranscriptional processing events requires a mechanistically description of the involved steps. The capping addition, which will be the first mRNA processing event to be analyzed here, happens in the nucleus as soon as the nascent transcript is 20 30 nucleotides long by a sequential action of three enzymes: RNA 5’triphosphatase, guanylyltransferase, and N7G-methyltransferase (16,17). Both, guanylyltransferase and N7G-methyltransferase bind to the phosphorylated C-terminal domain (CTD) of the RNAP II, which increases the efficacy of the capping process (18). After the addition of the 5’-end to the precursor mRNA, proteins get assembled on that structure such as the cap-binding complex (CBC20 and CBC80) (19) which protects the molecule from a 5’→ 3’ exonucleolytic degradation. Interesting, once the transcript is Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F exported to the cytoplasm, the CBC proteins bound to it are usually replaced by the 4E subunit of eukaryotic initiation factor (eIF4E) (17), promoting the ribosome attachment (20). In eukaryotes a second mechanistic approach to be considered in the context of premRNA processing and surveillance is the intron removal process (Figure 1). In mammals the majority of the genes go through this process. Three canonical splicing signals are required to remove most introns: a 5’ splice site (AG│GURAGU, where R = purine), a 3’ splice site (YAG│RNNN, where Y = pyrimidine and N = any base), and a branch point sequence located within 50 nucleotides upstream of the 3’ end of the intron. For splicing to occur, two consecutive transesterefication reactions take place that result in the fusion of two exons (21). In those events the small ribonucleoproteins (snRNPs) U1, U2, U4, U5, U6 and the Lsm proteins (Lsm 2-8) assemble together in a complex called spliceosome, which acts direct on the exon joining (22). Moreover, several studies have shown that the CBC proteins enhance the interaction of the snRNP U1 at the 5’ splice site (11,23), demonstrating the existence of interconnected mechanisms in the mRNA processing (21). In addition, conserved sequences characterized as positive-acting elements within the exons (the exonic splicing enhancers, ESE) are recognized by specific proteins such as those rich in serine and arginine (SR), whose interaction modulates the efficacy of the splicing mechanism. The cotranscriptional splicing processing is undoubtedly an important cellular tool that contributes to the final quality of the mRNA in several different organisms (23,24). In humans the mechanism seems to be extremely complex due to the extension length of some of their genes such as the dystrophin gene (14000 bp) (25). The huge introns must be precisely removed in order to maintaining a healthy organism. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F Following the discussion concerning the cotranscriptional modification of the precursor mRNA, RNA editing was ignored for a long time maybe due to the difficulties in identifying regions containing a single nucleotide edition (26). The enzymatic modification of nucleotide sequences was originally identified in the mitochondrial RNA of trypanosomes (27). Since then this mechanism of RNA processing has been extensively explored and described in a wide range of species. In mammals there are two main classes of editing enzymes. One of those classes is involved in the edition of cytidine (C) to uridine (U) in RNA molecules due to the activity of APOBEC-1 [apolipoprotein B (apo B) editing catalytic subunit 1] (28). In humans, proteins from the APOBEC family have being identified, and interestingly, most of them are involved in DNA edition of immunological system cells (29) that contribute to the adaptative immunity. A second class of those editing enzymes is responsible for the edition of adenine (A) to inosine (I); in humans adenosine is frequently edited to inosine by ADARs (adenosine deaminases that act on RNA) enzymes, whose activity is more intensive in the nervous system (30). The phylogenetic analyses of the two classes of proteins have indicated that they evolved from a common ancestral cytosine deaminase involved in pyrimidine metabolism (31). Finally, but not less important than the others cotranscriptional mRNA processing steps is the 3’-end formation (Figure 2). With the exception of some histone mRNAs, the eukaryotic mRNA possess poly(A) tails at their 3’-end, which are produced by a two-step reaction involving endonucleolytic cleavage and a subsequent poly(A) tail addition. In mammals the formation of the mRNA 3’-end requires the enzymes poly(A) polymerases (PAPs) plus four multimeric complexes: cleavage and polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CstF), cleavage factor I (CFI), and cleavage factor II Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F (CFII). CPSF and its subunits bind to the canonical polyadenylation signal AAUAAA, which is located upstream of the cleavage site (32). CstF, containing three subunits (CstF-50, CstF-64, and CstF-77), recognizes the so-called downstream U or G/U rich element of the cleavage site in the mRNA precursor (33). Working in a synchronous way, the complexes CPSF and CstF recruit the cleavage factors to the precise location of the cleavage. Poly(A) polymerase is usually required for the cleavage reaction and, together with CPSF, directs poly(A) addition. Poly(A) binding protein, PABP II, binds to the emerging poly(A) tail and in turn enhances the processivity of the poly(A) polymerase (34). Poly(A) tails usually reaches 200 nucleotides length in different mRNAs, and upon export to the cytoplasm, the nuclear PABP (PABPN1) that interacts with the poly(A) tails is replaced by the cytosolic poly(A)biding protein (PABPC) (35), which interacts with the translation initiation factor eIF4G that associates with eIF4E. This stimulates translation and regulates mRNA stability (36). All this discussion supports the existence of a complex and integrated network of cotranscriptional mRNA-processing events. Different inherited and acquired human diseases exemplify errors on such events, and from a medical perspective, either loss or gain of function can be relevant for maintaining a healthy organism. Therefore, the understanding of mechanistic hallmarks of mRNA processing and surveillance, highlights the relevance of these phenomena and illustrates the implication of them on the clinical diagnostics and therapeutic strategies. RNA surveillance: the molecular mechanisms that ensures mRNA quality control RNA performs multiple diverse functions in a cell, and therefore plays important functions in human diseases. Due to the interconnection of the cotranscriptional events, the Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F surveillance mechanisms are compounded by several distinct elements. In a cell, the exosome can be considered one important component of the surveillance system. The exosome machinery comprises a versatile complex of 3’→5’ exonucleases that degrade mRNA in both nucleus and cytoplasm (3,37,38,39). This complex is also absolutely required for processing small nuclear RNA (snRNA), ribosomal RNA (rRNA), and small nucleolar RNA (snoRNA) (40). Researchers studying yeast have demonstrated that normal or aberrant transcripts that extends its time in the nucleus become a target for degradation by the nuclear exosome (38,41). In the defective mRNAs, processing could take a longer time due to the defects in the body of the transcripts, which in turn can activate the nuclear RNA surveillance machinery. It is intriguing to consider the possibility that all RNA transcripts may be subject to the extensive surveillance mechanisms and mechanistic details concerning the RNA processing and degradation involved in the surveillance have been described. It is possible that all transcripts are potential substrates for the exosome, but those that are correctly processed interact with a bulk of proteins that protect them from exosome-mediate degradation (42). Researchers studying the human exosome components described that in patients with autoimmune scleroderma syndrome the PM-Scl75 and the PM-Scl100 exosomal subunits are recognized and destroyed by autoantibodies (37,43). This illustrates the importance of the exosome complex in organism integrity. However, the details concerning the exosome activity on distinguishing mRNA substrates and directing them to degradation remain to be investigated. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F The nonsense mediated decay (NMD): how can this mechanism work on quality control checking? Once the transcripts are synthesized and processed within the nucleus, they must be transported to the cytoplasm. In there, they have several potential fates and one of them is to become a target for the NMD. This process was initially described in Saccharomyces cerevisiae (44), and actually, it is known that one-third of inherited genetic disorders and many forms of cancers are caused by frameshift or nonsense mutations, which generate premature termination codons (PTCs). Contrary to intuition, the predominant consequence of nonsense mutations is not the synthesis of truncated proteins, whose deleterious effect could be crucial to the organism. The NMD surveillance mechanism accelerates the degradation process by its ability to bypass the rate-limiting step of deadenylation prior to decapping, and may perform a 5’→3’ mRNA decay (45) (Figure 3). Indeed, the phenotypic severity of selected diseases caused by nonsense-mutations can be predicted by the extent of reduction in the mRNA level of the mutant allele (5,7). Chain termination mutation reduces mRNA abundance by decreasing its half-life. For example, a recessive form of βthalassemia common in Mediterranean population results from a mutation that generates a premature UAG stop codon leading to a reduction of β-globin mRNA accumulation (8). Studying NMD, researches have demonstrated the existence of mechanistic particularities of this mRNA decaying process in different organisms. A combination of two cis elements is always required to trigger the process: an abnormality in the mRNA sequence, such as PTC, and a second RNA element such as a downstream sequence element (DSE) in yeast or an exon-exon junction in mammals. Particularly in mammals the NMD process is marked by the assemblage of many proteins, which form a complex known Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F as exon junction complex (EJC) (46). Those proteins are loaded on the transcript in the course of splicing at 20-24 nucleotides upstream of the exon-exon junction. In mammals the exon-exon junction is the cis-acting element that acts in conjunction with the PTC triggering NMD (47,48). Parallel PTCs are located at 50-55 nucleotides upstream from the exon-exon junction and are usually recognized by the Upf proteins (49). A current model suggests that during the first round of translation the relative position of the PTC from the EJC is recognized and the transcript is targeted for degradation. Studies on the EJC of Hela cells have demonstrated the existence of a central complex composed by REF/ALY, Y14, and MAGOH proteins (50). After the initial binding of those central proteins on the mRNA, Upfs are recruited and act on the induction of NMD in the presence of a PTC. In humans, the first medical significance of NMD was described in β-thalassemia that leads to β-globin mRNA degrading. Scientists now known that several others diseases can be explained by NMD including brachydactyly type B (51), von Willebrand disease (52), factor X deficiency (53), and retinal degeneration (54). Those and several other diseases mediated by NMD demonstrate the importance of the surveillance mechanisms in protecting the organism against aberrant protein forms. Moving further: microRNA mediating the post-transcriptional processing MicroRNAs (miRNA) are little endogenous non-coding mRNAs of about 2123 nucleotides that control fundamental cellular process in a variety of organisms. Since their discovery in the 1990s a multitude of basic information has been accumulated, which has identified their function in post-transcriptional control, either via degradation or via translational inhibition of target mRNAs. They fine-tune gene expression, working in parallel Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F with transcriptional regulatory processes (55,56). In the current model, miRNAs are initially formed by long precursors known as pri-miRNAs (57,58), which are processed in the nucleus by Drosha. This process forms double stranded fragments that are export to the cytoplasm and in there they are further processed by a second RNAse type III endonuclease, i.e., Dicer. This enzyme produces dsRNAs, which contain 21-22 nucleotide fragments, paired in the 19 central nucleotides with 2 overhang ends (59). These small molecules are characterized as mature miRNAs and can be subsequently incorporated into a complex termed RNA-induced silencing complex (RISC). A miRNA incorporated into RISC is able to guide the RNA interference (RNAi) machinery to its target and complementary mRNAs by forming RNA duplexes, which result in sequence specific translation repression (60,61) or mRNA decay (55,62,63). Interesting, computational analyses suggests that 20-30 % of protein-coding genes are likely targets of miRNAs (64), suggesting the importance of those molecules in the surveillance mechanism due to their ability to degrade the mRNA when it is claimed by the quality control mechanism. miRNAs expression profile are highly dynamic during embryonic development as well as in adulthood. The misexpression of miRNAs can disrupt embryogenesis and tissue homeostasis. Moreover, evidence from gainand lossof function studies indicates roles for miRNAs in pathophysiologic states including cardiac hypertrophy, muscle dystrophy, hepatitis infection, diabetes, Parkinson disease, hematological malignancies, and several types of cancers, but the miRNA mechanistic processes in diseases are still largely unknown. Preliminary studies concerning the therapeutic potential of miRNA and the RNAi processes were initially done in 2001 (65) and are currently being further developed. The use of miRNA is still a promising therapeutic strategy, but a clearer understanding of the Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F mechanistic details mediated by miRNA and how they fit into the quality control mechanisms need to be clarified. Moreover, the establishment of an efficient system to deliver those small RNAs to the desired tissue is considered the biggest challenge to their clinical application (66); therefore, many efforts have been performed on that approach. The cis and trans elements working on the mRNA surveillance: how do they interconnect the story? mRNA turnover helps the cell to reach its own biochemical equilibrium. In mammalian cells, this mechanism requires a precise interconnection of physiological signals (5,67). The degradation rate of a specific transcript is always modulated by both cisacting elements within the mRNA body sequence and trans-acting factors that bind to the first (67,68). In the cis-acting elements, sequences rich in A and U nucleotides also called AU-rich elements (AREs) have been demonstrated to be involved in the mRNA rapid degradation. Those elements are regularly found in the 3 ́-UTR (untranslated region) of several short half-life mRNAs such as those that encode cytokines, growth factors, and oncogenic proteins (69,70,71). Frequently, those elements are recognized by specific AREbinding proteins (AREBPs) that modulate the kinetic rates of mRNA degradation. For the last two decades, researchers have been conducting studies to clarify mechanistic details of ARE and AREBPs in mediating mRNA processing. Many of those results have demonstrated a direct correlation between an unbalance mRNA turnover of ARE-containing mRNAs and the development of cancer, inflammatory processes, arthritis, and other organic dysfunction (72,73). Amongst the broadly studied AREBP, HUR and TTP are example to be considered next. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F HUR is a member of a super family of elav-related proteins, which is differentially expressed along the embryonic development (74). It has a predominantly nuclear localization and can shuttle between nucleus and cytoplasm (75), serving as an adaptor for the nuclear export of some mRNA containing ARE (76) and contributing to the surveillance process mechanism in a cell. Besides, working as a trans element, this AREBP contributes to the mRNA stability due to its binding (77,78) and in an unbalanced environment HUR can be directly involved in the development of cancers such as colorectal carcinoma due to cyclin mRNA stabilization (79). On the other hand, TTP or tristetrapolin protein is a predominant cytoplasmic molecule and TTP-binding can cause degradation of the target mRNA. Containing two classical zinc fingers, this protein binds to mRNAs classified as class II ARE such as TNFα, GMCSF, and IL-3 (80,81). Research conducted with transgenic mice, where the TTP gene was previously knocked down, demonstrated an increased level of TNFα protein and consequently the mice developed several clinical symptoms such as erosive arthritis, dermatitis, and myeloid hyperplasia (82). This study suggested the relevance of TTP in the control of the development of several clinical diseases, which are mediated by the action of the protein in the regulation of mRNA level of several important molecules. Based on the above observations, mammalian cells are able to interconnect cis and trans elements in order to modulate the rate of mRNA turnover and to regulate important physiological and pathophysiological functions. Myotonic distrophy and several other diseases, which are related to the mRNA metabolism, will be considered in the following sections. The majority of them demonstrate the existence of a broken balance between the elements involved in the mRNA turnover events. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F How can a cell assure the RNA surveillance? A kinetic energetic conformational based model To assure homeostatic steady-state in a cell, aberrant RNAs must be distinguished from normal ones and rapidly degraded by the cellular machinery. In all living organisms, the efficiency and the accuracy of such biochemical mechanisms are the result of billions of years of evolution. Particularly in eukaryotes, mRNA undergoes maturation through several steps and it seems imperative that the mRNA surveillance process occurs in parallel to each of those steps. As previously described, mRNA molecules play a key role in cellular biology, and in order to fulfill their functions, they need to fold into complex tertiary structures. Considering the dynamic biological system in a cell, mRNA folding happens cotranscriptionally and is able to influence the general folding process of the mature mRNA molecule, either positively or negatively, which either prevents or favors the generation of non-native trapped intermediates (83), impacting on the general RNA maturation process and cellular homeostasis. During transcription RNA molecules are dynamically refolded several times and surveillance can help in the quality control of those transcripts. However, how can the surveillance mRNA machinery efficiently differentiate the corrected and wellprocessed molecule from the aberrant ones in the cellular complex environment? In this section, a model that might be able to safeguard the surveillance mechanism in a cell, helping on the maintenance of the complexity of life, will be discussed. Since the initial X-ray diffraction studies of biological molecules performed in the first decades of the twentieth century, scientists have been interested in understand how the molecular structures can explain the biological function of the molecules. Nowadays, X-ray Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F diffraction, nuclear magnetic resonance, computer structure prediction based on sequence homology, and other techniques have been helping researchers to elucidate the molecular structure of biological compounds and, as a result, pharmacological agents have been developed to target specific regions of molecules, which facilitate the mechanistic action of the drugs. However, the knowledge of the tridimensional structure of such molecules by no means constitutes the full information about their biological activity. In general, large molecules in a solution are not rigid (84) and during their life cycle in a cell, biological molecules can actively interact amongst themselves by forming functional complexes that require conformational changes of the constituents, as seen in the components of the mRNA surveillance machinery. Based on those two observations, one can conclude that the dynamic of the conformational rearrangements and, consequently, the thermodynamic significantly contribute to building molecular models for every step of the gene expression process in a kinetic energetic conformational model. Moreover, several studies have demonstrated that the overall stability of complex interactions are dictated by standard Gibbs free energy change (ΔG°), which involves both enthalpic and entropic contributions, giving the thermodynamic parameters an important position in the study of molecular interactions and their functions in a cell (84,85,86,87). Regarding the mRNA surveillance, sets of protein complexes are required all along the pathway. These proteins, working in an orchestrated way, follow the rules of the thermodynamics in assembling to their substratum. As in most biological process, mRNA surveillance has a higher degree of accuracy in its mechanistic procedure than could be reasonable explained by the kinetic proofreading processes. First proposed by John Hopfield (1974) (88) and next by Thompsom and Stone (1977) (89), the kinetic Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F proofreading model is an established and acceptable mechanism that explains the accuracy of most of biological reactions, including those involving nucleic acid interactions (88,90,91,92). In the proposed model, the highly selective recognitions in molecular reactions are carried out enzymatically and are strongly driven by the hydrolysis of nucleoside triphosphates. The mechanisms happen in two distinct irreversible steps, which increase the specificity of the interactions between molecules that are conducted by the difference in free energy. Interestingly, the kinetic proofreading model involves an intermediated product formation. In the initial step of the reactions, non-cognate molecules are rejected, but near-cognate ones could be involved in the binding reactions between two molecules. During the second step of the reactions after the nucleoside triphosphates hydrolysis, the kinetic proofreading model allows a time delay in order for the equilibrium in the reaction system to be reached. This delay makes it possible to discriminate between the correct and the incorrect substrate based on the energy differences between them, which increase the dissociation rate of the non-cognates substrates for the binding and in some circumstances RNA degradation. The second step of the kinetic proofreading reactions expends energy, but minimizes the binding of incorrect molecules in the reaction. This step has a fine-tuning approach to the effectiveness of the biological reactions in a cell that explore the general fold of the molecules and their thermodynamic interactions. In the surveillance process, where a series of concatenated reactions are performed and the RNA is matured and processed, a minor error along the pathway could be disastrous to cellular homeostasis. The kinetic proofreading provides a potential rationale for the complexity and efficacy of the mechanism. Along with the correct molecular interactions mediated by hydrogen bonding, salt bridges, π-π and π-cations stacking Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F interactions, van der Waals contacts, plus hydrophobic interactions of aliphatic molecular parts (84) between the mRNA surveillance machinery and the ribonucleic acid, the overall stability requires a thermodynamic approach in order to distinguish the correct binding without mistakes. Those incorrect molecules facilitate neither a correct molecular interactions with the surveillance proteins nor a correct thermodynamic event, which favors the identification of the mistakes on the mRNA molecule by the kinetic proofreading mechanisms. Once the errors are identified, they can lead to the mRNA body degradation, which safeguards a healthy life. Looking forward the clinical approaches The quality control in mRNA biogenesis: if it goes bad, how can it influence human clinical diseases? The regulation of gene expression occurs at multiple stages and, as previously discussed, transcription and RNA processing have major functions in the quality control mechanisms of an organism (Figure 4). So far, a lot of effort has been directed to the understanding of how errors in the RNA processing could be related to the development of diseases. From the medical point of view, biomarkers have been investigated in order to help diagnose important clinical diseases such as those related to the mutations in the 20210G>A (F220210*A) of prothrombin gene (93). This mutation raises prothrombin plasma concentrations and predisposes carriers to develop thromboses (94). Next, this review will briefly describe examples of defects in RNA processing mechanisms and how the RNA quality control machinery works in the surveillance process in a cell. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F Mutations in sequence conserved elements Mutations can be potentially harmful to an organism, if transcripts containing such aberrant sequences are not recognized by the mRNA surveillance machinery. Several studies have been performed on the globin mRNA related to mutations and their side effects on an organism. Globins were the first human genes to be cloned (95), and represent the earliest medically important genes that illustrated how their expression pathway can be inactivated by naturally occurring mutations. The remarkable phenotypic diversity reflects the heterogeneity of mutations on the globin loci (96) Of the globin diseases, thalassemias is one of the best characterized, and accomplishes several molecular abnormalities caused by punctual mutations on α or βglobin genes. Thalassemic patients have a variety of symptoms determined by hemolysis and ineffective erythropoiesis, which can result in a complete lack of symptoms to a sever-transfusion dependent anemia (97). Based on the investigation of the 3’-UTR of globin mRNAs from thalassemic patients, different mutations on the canonical AAUAAA polyadenylation signal of both α -globin (98) and β-globin genes (99) were described. Interesting, the different types of mutations found demonstrated a clear ethnic distribution that correlated to the existence of a founder effect and the natural selection that in some cases works positively on the side of the heterozygote. Besides the globin mRNA analysis, other studies involving mutation in the canonical AAUAAA region have also been performed. Between the investigations, mutations in the AAUAAA region of the Foxp3 mRNA were identified and shown to be related to the development of IPEX syndrome (100), a rare and fatal disorder characterized by Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F polyendocrinopathy, enteropathy, and immune system dysfunction. Fabry’s disease is another clinical manifestation due to a severe X-linked recessive inborn error of glycosphingolipid catabolism, which results from a mutation on the lysosomal alphagalactosidase A (alpha-Gal A) gene. The alpha-Gal A is a typical gene that bears its polyadenylation signal within the coding region and the gene lacks the 3’-UTR (101). The deletion of an AA dinucleotide on the internalized poly(A) site leads to an aberrant 3’-end. This provides a generation of several different non-functional transcripts and a complete inactivation of the gene. Furthermore, other diseases can be taken as examples to illustrate mutations on the RNA conserved poly (A) signal AAUAAA: the acetyltransferase 1 polymorphism in colorectal cancer (102), metachromatic leukodystrophies (103), X-linked sever combined immunodeficiency (104) etc. These examples clarify the functional importance of the mRNA conserved poly(A) region in keeping an equilibrated organism. However, regardless of the kind of mutation, most of them may alter the quality and level of the protein that will be translated and a mutation does not necessarily abolish the production of the final product. But this unbalance in the cellular equilibrium is able to initiate an illness process. Remarkable studies have been developed to describe and understand the influence of other mutations on the mRNA processes and their clinical relevance. Several mRNA cisacting elements have been investigated and clinical relevant mutations found. However, a couple of them need more investigation concerning their biochemical and phenotypical effects on an organism. Many of those elements are directly correlated with the 3’-end processing of the mRNAs and between them a mutation in the cleavage mRNA site of the prothrombin (coagulation factor II; F2) (105) was described. The cleavage mRNA region is Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F localized 10 to 30 nucleotides upstream of the polyadenylation site and in a mutated F2 gene there is a CA → CG transition that is responsible for raising the thrombin plasma concentration. This increment in the protein level disturb the finely tuned balance between proand anti-coagulatory activities, resulting in an increased risk to develop trombophilia (106), which can be characterized by several endothelial injuries and hypercoagulation disorder. Subsequently other mutations on F2 gene were identified and all of them are related to the mRNA cleavage site at the 3’UTR, leading to an unbalanced regulation of gene expression. Investigations in the RNA processing field have pointed out different elements that work in an integrated network in the context of the mRNA surveillance steps to monitor mRNA structure and function (1,11). Medical perspectives on applying such knowledge for therapeutic procedures is in high demand, however, the difficulty resides in the identification of a common mechanistic pathway or a key biochemical element that works on the recognition and on the surveillance of such aberrant molecules. Based on different studies, scientists have realized that the mechanistic processes related to the mRNA surveillance in a cell have evolved independently, possibly in parallel with the evolution and complexity of the genome structures and transcript-processing pathways (107). A more detailed understanding of mRNA surveillance systems is therefore likely to reveal previously unknown determinants of gene regulation and this will facilitate a clinical approach against the defects in the surveillance mechanisms. mRNA metabolism and cancer Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F Cancer is the most serious clinical manifestation in our modern world. In a cancer cell several mutations are present in its nuclear material, but in order for the disease to start developing intracellular changes must be modulated by extracellular signals that turn on the oncogenic machinery. In an attempt to find key elements that lead to cancer, signal transduction pathways were elucidated and a correlation between errors on the mRNA metabolism and uncontrolled cell growing was found (108,109,110). A strict regulation of mRNA level is absolutely required for the maintenance of the cellular steady-state and in a cancer cell the mRNA turnover is frequently altered. Transcripts that usually have a 30 minutes half-life, such as the mRNAs of protooncogenes and cytokines, which contain the cis-acting ARE at the 3’-UTR, have a significant increase in their half-lives in cancer cells. In the classic studies, Hollis et al. (1998) (111) demonstrated in myeloma plasma cells a seven-times increase in the c-myc mRNA halflife, due to a chromosomal translocation that disrupts the ARE region at the 3 ́-UTR of the transcript. Interesting, the same effect of chromosomal translocation on the c-myc gene is found in Burkitt ́s lymphoma (112). Other defects on mRNA metabolism can also lead to serious health problems such as those related to the alternative splicing of the CD44 glycoprotein mRNA. CD44 protein is characterized as an adhesion cellular molecule involved in cell-cell interaction, cell adhesion, and migration and is also characterized as a receptor for hyaluronic acid and other ligands such as collagens and matrix metalloproteinases, which allow this protein to participate in a wide variety of cellular functions (113,114). The alternative mRNA splicing is the basis for this functional diversity of the CD44 proteins, and studies have demonstrated that this protein may be related to tumor metastasis, since different isoforms where identified in some types of tumors such as Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F lymphomas (115) as well as cervical 116), breast, and prostate cancer cells (117). However, what controls the production of the CD44 problematic isoforms mRNAs and how they evade the surveillance machinery in a cell is still not understood. Furthermore, a study on the transcriptional machinery in certain human cancers such as ovary, colon, pancreas, and breast cancer (118) detected an overexpression of PAP, which favors its use as a molecular biomarker in certain kinds of cancers (119,120). Other types of biomarkers are currently being investigated in order to provide a powerful tool for the clinical diagnosis of this problematic threat. From a medical perspective, there needs to be more investigation in order to explain all the mechanistic details in a cancer cell. Once, more details are clarified, more therapeutic decisions could be taken in a strict and realistic way. Differential mRNA processing in human diseases and RNA pathogenesis as the focus of medical research Several neurodegenerative diseases are also caused by mistakes in the mRNA processing steps, due to an exaggerated nucleotide repetition in the mRNA sequencing. Those expansions usually originate during mitosis and/ or meiosis and in general are present in a normal cell; however, in a pathogenic condition, the number of nucleotide repeats is considerably high, turning the transcript into a toxic molecule to the cell. Fragile X Mental Retardation (FMR) (121) and the Spinal and Bulbar Muscular Atrophy (SBMA) (122) were the first two of those pathogenies identified in 1981. FMR disease is an inherited mental impairment that can range from learning disabilities to severe cognitive and intellectual disabilities. In this disease there is a trinucleotide (CGG) repetition at the 5 ́Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F UTR of the FMR1 gene; in a normal person this gene contains less than 44 trinucleotide repeats; however, patients have a hyperexpansion of a polymorphic CGG repeat in the gene. Expansions of 55-200 repeats are called premutations and characterize carriers usually have no mental impairment. A full mutation exceeds 200 CGG hypermethylated repeats, which leads to a transcriptional silencing of the gene and absence of the Fragile X mental retardation protein (FMRP). Due to the considerable number of patients, diagnostic methodologies have been developed involving molecular and immunocytochemical approaches. The other clinical condition initially reported is the Spinal and Bulbar Muscular Atrophy, which is also an X-linked threat that causes degeneration of motor neurons, with adult onset and slow progression. The main symptoms are weakness and atrophy of bulbar, facial and limb muscles, but sensory disturbances are frequently found in SBMA patients. This disease, like the previous one, involves hyperexpansion of trinucleotide repeats (CAG) in the coding region of the androgen receptor (AR) gene and the length of expansions is correlated with the intensity of the disease symptomatology. Patients with longer CAG repeats (≥ 47) will present a more accentuated clinical phenotype than patients with shorter expansion. In any case, the problematic transcript is not recognized by the quality control machinery and leads to the production of a toxic molecule protein that curiously aggregates inside the cell. Since the initial studies, several pathogenies due to trinucleotide repeats expansion have been described such as Huntington ́s disease (HD) (123,124), spinocerebellar ataxias (125), myotonic dystrophy (126), and many others. Interesting most of those diseases have a founder effect (127) and several studies have suggested that new mutations occur in a Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F subgroup of unstable high-normal triplet repeat alleles with a particular founder chromosomal haplotype (124,128). The trinucleotide repeat disorders are considered a serious health problem that has been demanding many efforts on the clarifying their molecular mechanisms. Among all the clinical manifestations due to the nucleotides expansion, some muscular diseases require special attention due to their differential level of clinical manifestation, including life impairment. Myotonic dystrophy (DM), also known as Steiner’s disease, has been broadly investigated. It is a progressive disease characterized as a multisystemic disorder whose patients develop skeletal muscle weakness, myotonia, cardiac conduction defects, dilated cardiomyopathy, endocrynopathy, alteration in smooth muscle function, and cognitive impairment (129). DM is an autosomal dominant trait and two genetic loci presenting nucleotide repeats expansion have been associated with the disease phenotype; DM1 in the chromosome 19q13.2-q13.3 and DM2 in the chromosome 3q13.3-q24. DM1 is caused by an expanded (CUG)n repeats in the 3’-UTR of the myotonic dystrophy protein kinase (DMPK) gene (130,131). Like other trinucleotide disorders expansion, an unaffected individual has from 5 to 40 repeats. Patients with 41-180 repeats have mild symptoms, and those presenting repeat sizes > 1500 result in congenital DM, a severe form of the disease characterized by mental retardation, hypotonia, and symptoms associated with severe muscle weakness. Many congenital patients die soon after birth from respiratory problems due to underdeveloped diaphragm and intercostals muscles (129). The mechanism that a non-translated CUG repeat in a single allele results in the severe dominant phenotype of DM1 remains unclear. One possible explanation is that this series of repeats in the 3’-UTR of the DMPK gene may disrupt the expression of other genes at either the DNA or RNA Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F level (132). Maybe, the triplet expansion can be bound in a competitive way by proteins that are directly involved with the mRNA processing which affects the expression of a number of genes by a trans-dominant effect (133). DM2, on the other hand, closely mimics the phenotype of adult-onset DM1, but DM2 is not associated with severely atrophic facial and forearm muscles. Myotonic dystrophy type 2 is due to the (CCUG)n nucleotides in intron 1 of the zinc finger 9 gene (134) and as in the case of DM1, studies suggest that the disease is correlated with the disruption of certain kind of molecular signalization pathway and the direct involvement of CUG-binding proteins have been reported. Special attention has been giving to the CUG-BP isoforms as a key mediator of the pathogenic effect of myotonic dystrophy disease (135), due to its direct influence on the mRNA metabolism (136,137,138). More biochemical mechanistic investigation must be performed in order to develop some clinical palliative treatment for the patients. Those and several other pathologies represent a broken balance in the cellular mRNA metabolism that is really important in the context of a healthy life and RNA pathogenesis has become more the focus of medical research. Furthermore, as mention before, considering the complexity of the mRNA surveillance processes, it is extremely important to keep developing clinical and methodological approaches for detecting changes in the surveillance system in a pathological condition. Several laboratories have been trying to improve their strategies on those approaches, and an interdisciplinary cooperation and the use of up-to-date methods are required such as DNA amplification and sequencing, microarrays, mass spectrometry, as well as several other modern techniques currently applied in molecular biology laboratories. As a result, strategies to identify disease genes that correlates with failures in the RNA surveillance machinery have been developed Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F (139,140,141). However, in spite of all the research being conducted in the field, scientists do not yet understand all the details, for example, what directs the recruitment of NMD factors at the premature termination site (142). Details like this has a negatively impact on the development of a universal methodological view to detect pathologies due to mechanistic problems in the RNA surveillance machinery. Maybe in a near future, a particular and unique kinase mediating mRNA processing and surveillance will be identified and a sophisticated diagnostic procedure able to differentiated normal from aberrant RNA surveillance mechanisms will be developed. This is suggested based on the knowledge that the phosphorylation mechanism has been identified as an important factor in the posttranscription quality control of the mRNA (143,144). For while, the mRNA surveillance is still an enigmatic and intricate puzzle for molecular biologists, human geneticists, and medical community. Modern therapeutic strategies for RNA-based diseases mRNA target based therapy is an emerging and powerful alternative for the treatment of genetic disorders due to mutations or RNA processing defects that directly correlate with the RNA surveillance mechanisms. Although an emerging field, RNA therapy has the potential to modify native mRNA transcripts within a normal regulatory environment. The approaches range from complete degradation of specific mRNA targets to modification of mature mRNA molecules. The knowledge of the structure and function of RNA biology is increasing the development of the RNA-based therapy strategies. Many of the effector molecules underpinning these novel methods have their origins in natural biochemical pathways that have been discovered in the recent years. Since the approval of Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F the first RNA-based drug for the treatment of a human disease by the US Food and Drug Administration (FDA) in 1998, there has been an increasing biotech company gold rush for patents on an efficient drug deliveries system to the desired tissues. Next, two major RNAbased strategies to treat human diseases are briefly described. Antisense oligonucleotides (AO): Initially designed to down-regulate gene transcription, AO are currently been use to bind a complementary sequence in a target pre-mRNA by altering its processing. Specifically, AO are currently being employed to block sequences in a target mRNA that are critical for its splicing events to avoid the production of undesired disease-causing mRNA product. The synthetic molecules have been used in the studies of therapeutical strategies for the treatment of β-globin, β-thalassemia, Hutchinson-Gilford progeria syndrome (HGPS), Duchenne Muscular Dystrophy (DMD), Spinal Muscular Atrophy (SMA), Amyotrophic Lateral Sclerosis (ALS) etc (145,146). Human clinical trials have been performed and several drugs are currently available for the treatment of human diseases such as formivirsen, an AO that blocks the synthesis of important proteins of cytomegalovirus, which are responsible for certain kind of human eye inflammation; mipomersen, an antisense RNA drug that reduces the production of Apo-100 in patients with hypercholesterolemia etc (147). However, re-administration of the AO may be necessary, considering that those compounds have a limited biological half-life in targeting transiently the transcription and/ or mRNA processing. To eliminate this problem, recombinant adeno-associated viruses (AAV) have been used as delivery vehicles to transduce antisense RNAs to the target cells (148). However, problems such as tissueMolecular Medicine www.molmed.org U N C O R R E C TE D P R O O F specific targeting, toxicity, and immune response to the viral vectors still pose a problem in correcting the RNA errors and their surveillance machinery. RNA interference: Based on the natural characteristics of miRNAs that are able to interfere in posttranscriptional control either via degradation or translation of targets mRNA, the RNA interference (RNAi) mechanisms have been studied and improved as a promising therapeutic method for knocking down genes whose activity correlates with pathogenesis. The key effector molecules of the RNAi are 21-23 ribonucleotides length and are able to target a unique mRNA sequence or even specific splice variants (149) and as a consequence to decrease the mRNA expression. RNAi-based therapy has been used in clinical trials for the treatment of HIV (150), hepatitis B virus (HBV) (151), macular degeneration (152), Alzheimer disease (153), liver cancer (154) and others. Moreover, the specificity of the RNAi also opens the possibility of targeting specific mutant alleles associated with dominant genetic diseases (155). However, while RNAi offers a novel therapeutic strategy for several diseases, the delivery of such molecules is still the major hurdle for their clinical use as a regular pharmaceutical agent. The non-specific off-target sequence recognition for the small 21-23 ribonucleotides, continue to be a significant concern (156). One the other hand, the historic success of small molecules as a pharmacological agent makes them attractive for use in therapeutic trials (146). There needs to be more investigation on mechanistic particularities of the RNA surveillance pathways in order to progress with RNA-based therapy strategies. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F Connecting the whole tale: mRNA surveillance under different cellular environmental conditions. How is it possible to assure homeostasis? Many behavioral, physiological, and biochemical actives in a variety of organisms have been reported to show a circadian rhythm driven by endogenous biological clocks. Circadian rhythms allow organisms to coordinate their physiology with day-night cycles and environmental changes, and may have first evolved to control cellular metabolism (157). To date, studies have focused on the understanding of the molecular mechanisms that underlie those rhythmic clocks and genetic analyses have identified numerous clock genes in different species. Across evolution, the molecular circadian clock is self-sustained and typically consists of auto-regulatory loops regulated by specific proteins that are rhythmically translated in an integrated manner (158), and transcription is considered the most critical step in the control of such circadian rhythms (159,160). In mammals, the core molecular clock components are: period genes, Per (Per1, Per2, and Per3), cryptochrome genes, Cry (Cry1 and Cry2), two helix-loop-helix transcription factors (Clock and Bmal1 genes), casein kinase I epsilon (Csnk1e), and two nuclear hormone receptor genes (RevErbAa and Rora). Other clock-controlled genes like the transcription factor Dbp and Nfil3 have also been identified (159,161). The basic mechanism of the circadian rhythm consists of the transcriptional activation of Per and Cry genes by CLOCK/BMAL1 heterodimers. Then the increased concentration of PER and CRY blocks the transcription regulated by CLOCK/BMAL1, which closes the auto-regulatory feedback loop (162). Thus, the balance between transcription and translation of certain genes keep the core clock Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F strictly regulated and remarkably plastic, which supports the circadian rhythmicity (163,164). In mammals, the suprachiasmatic nucleus (SCN) is considered the center of circadian clock in the body (162), which coordinates peripheral oscillators through neural and endocrine pathways (165). Circadian oscillators have been identified in peripheral tissues such as the heart (166), liver (167), bone marrow (168), endocrine tissues (169), pancreas (170) etc, and they must respond promptly to an initial stimuli captured by the SCN. A tight and precise control of the circadian rhythms in an organism assures its homeostasis; alterations in such rhythms can lead to serious pathologies such as sleep disorders, cardiovascular diseases, depression, and even cancer (171,172,173). However, in order for a body to interconnect all molecular signals generated by an initial stimuli, in every single cell the transcriptional, translational, and surveillance machineries take major responsibilities. As previously discussed in this review, the amount of mRNA in a cell is regulated at many levels; in this context mRNA processing and surveillance significantly contribute to changes in gene-expression patterns in response to external stimuli. More recently, experiments to determine circadian transcription on a large scale are currently being performed in several labs (174). Some evidence indicates that only a few and particular mRNAs present a degradation pattern in the form of a circadian rhythm (175,176) in different organisms despite the rhythmic increase in some protein level, which suggests an alternative control of the post-transcriptional and post-translational mechanisms. Several investigations point to a direct involvement of signaling cascades of protein phosphorylation being directly involved along the course of the day (177) that either stabilize a required protein for cellular mechanistically adjustments to the circadian clock or stabilize proteins Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F that are able to bind mRNA molecules, avoiding their premature degradation and making them available for more translational rounds. MicroRNAs were also identified as molecular elements that help in the regulation of the circadian machine (178,179). Due to the complexity of the circadian rhythms and their direct connection to the quality control of gene expression, scientists seek to reveal details in the circadian clock in order to understand how such events modulate the homeostasis of an organism. Actually, there is growing evidence that circadian rhythms are connected with the physiological state of an organism such as its nutritional condition, hormonal fluctuation, aging, and even diseases. These physiological conditions act positively or negatively on biological clocks but the events must be integrated to maintain the organism, however the genetic background of an individual supports the plasticity of the molecular circadian mechanisms. Rudic et al (2004) (180) demonstrated an impaired insulin responsiveness and reduced gluconeogenesis in Bmal1 -/mice; other studies have shown that those mutant mice present early aging and reduced lifespan. Those mutants also have a high accumulation of reactive oxygen species (ROS), supporting the idea that the circadian protein BMAL1 may participate in the oxidative stress responses (181) frequently observed in age-related neurodegenerative diseases such as Alzheimer and Parkinson (182). Maybe failures in the transcription and/ or in the surveillance machinery block the correct expression of bmal1, which favors the development of those neurodegenerative diseases. This corroborates the observation that the circadian clock must be tightly regulated in order to modulate a dynamic homeostasis in the body to assure the maintenance of life. In addition, more and more studies have demonstrated that the outside signals are captured by the SCN in mammals’ body. These signals are decoded as molecular Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F messages that regulate the integrity of the organism and its surveillance at the cellular and molecular level. As examples of such integrity, investigations have demonstrated that the levels of the metabolic hormones glucagon, insulin, ghrelin, leptin, and corticosterone oscillate in a circadian rhythm as oscilates the level of the peroxisome proliferatorsactivated receptor-γ (PPARγ) coactivator-1α (PGC-1α), which is an essential activator of gluconeogenesis in the nutritional deprived state (183,184,185). The molecular interplay between the circadian clock regulators mediate the circadian oscillation of those important functional molecules and fine-tune the final amount of every single metabolite in a cell. Transcription absolutely turns out to be a central mediator and along with its surveillance machinery assure a correct temporal and physical amount of mRNA molecules during the circadian rhythms. Once available, those mRNA molecules can be translated when required, contributing to the body’s homeostasis maintenance. Therefore understanding the details of this molecular fine-tune regulation of the circadian clock will provide a lot of information and insights about how to deal with pathological conditions related to disturbances in those circadian rhythms. Perspective Studies of the RNA surveillance mechanisms in eukaryotes are constantly reshaping our thinking about how errors in gene expression are detected and how they are controlled at coand post-transcriptional level. Eventually the growing knowledge of those processes is likely to have an important impact on clinical medicine as the era of genetic intervention develops. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F The regulation of mRNA processing and stability are nature’s intriguing and very effective molecular adaptation mechanisms, which enable a cell to maintain a translatable transcript upon all the energy-demanding steps of mRNA quality control survey. Moreover, the intricate mRNA quality control mechanism permits a cell to respond rapidly to changes in intrinsic and extrinsic stimuli, maintaining an equilibrated cellular environment. Failures in such molecular quality control mechanisms usually lead to the development of diseases. Considering the medical and clinical relevance of the mRNA metabolism, the 3’-UTR has clearly emerged as a unique region that controls important cellular function such as morphogenesis, metabolism, cell proliferation, and apoptosis. Actually, several ongoing studies are trying to identify novel sequence elements in the 3’-UTR that can modulate the mRNA surveillance processes. Unfortunately, many details concerning the mechanism of the mRNA quality control are still undiscovered, and need to be clarified in order to combine most of the information and develop therapeutic strategies. In addition, future research addressing the key changes in mRNP composition at each critical remodeling step of an mRNA, as it goes on its journey from the nucleus to the cytoplasm, will be crucial for understanding how mRNA decay, translation, RNA quality control, and circadian rhythms are regulated through interplay of different mechanisms. Currently, based on all the efforts of this field of investigation, there is a general expectation that in the near future molecular biologists, human geneticists, and the medical community will incorporate the new knowledge of the mRNA processing and surveillance mechanisms in the design of novel and realistic approaches to clinical interventions for a variety of abnormalities. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F AcknowledgementsI am in debt to Cristina Pacheco Soares and Newton Soares da Silva for all theassistance and friendship at UNIVAP. 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