Gene expression is controlled through a complex interplay among mRNAs, non-coding RNAs and RNA-binding proteins (RBPs), which all assemble along with other RNA-associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA-binding domains. However, various methods have been recently d...

RNA interference (RNAi) is a gene silencing mechanism that acts at both the posttranscriptional and transcriptional levels. We have recently identified an RNA-containing complex, named RNA-induced transcriptional silencing (RITS), that directly links RNAi to transcriptional gene silencing in Schizosaccharomyces pombe. Here we review the affinity purification methods we use to isolate RITS and d...

A procedure has been developed for the sequential removal and purification of the glycoprotein and membrane protein of vesicular stomatitis virus (VSV). Neither of these proteins exhibited transcriptase activity. All of the activity was recovered in the ribonucleic acid (RNA)-ribonucleoprotein complex of VSV, which also has four other minor proteins associated with it. During transcription of 4...

Recent studies have uncovered an unanticipated diversity of noncoding RNAs (ncRNAs), although these studies provide limited insight into their biological significance. Numerous general methods for identification and characterization of protein interactions have been developed, but similar approaches for characterizing cellular ncRNA interactions are lacking. Here we describe RNA Affinity in Tan...

Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific R...

We have devised a new procedure for the purification of highly active preparations of Bacillus subtilis RNA polymerase holoenzyme. A column of heparin-agarose A-15m is used to rapidly and quantitatively adsorb RNA polymerase from the initial crude extract fraction. This affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid s...

A new method for rapid purification and structural analysis of oligoribonucleotides of 19 and 20 nt is applied to RNA hairpins SL3 and SL2, which are stable secondary structures present on the psi recognition element of HIV-1. This approach uses ion-pairing reversed-phase liquid chromatography (IP-RPLC) to achieve the separation of the stem-loop from the transcription mix. Evidence is presented...