oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. Here we show at the ultrastructural level that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and mi...

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent bou...

In 1968 the Nomura lab in Madison made an astonishing observation: when ribosomal proteins were mixed under appropriate conditions with 16S rRNA, fully functional ribosomal 30S subunits were formed. Along with the subsequent total reconstitution of 50S subunits, it was established that even entire ribosomes could be assembled in vitro from their constituents. Neither energy in the form of nucle...

Long INterspersed Elements (LINE-1s, L1s) are responsible for over one million retrotransposon insertions and 8000 processed pseudogenes (PPs) in the human genome. An active L1 encodes two proteins (ORF1p and ORF2p) that bind with L1 RNA and form L1-ribonucleoprotein particles (RNPs). Although it is believed that the RNA-binding property of ORF1p is critical to recruit other mobile RNAs to the ...

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure ...

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 techn...

The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechani...

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners,...

Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the ...