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Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with(More)
HIV-1 Rev protein directs nuclear export of pre-mRNAs and mRNAs containing its binding site, the Rev response element (RRE). To define how Rev acts, we used conjugates between bovine serum albumin (BSA) and peptides comprising the Rev activation domain (BSA-R). BSA-R inhibited Rev-mediated nuclear RNA export, whereas a mutant activation domain peptide(More)
Ribonucleoproteins (RNPs) mediate key cellular functions such as gene expression and its regulation. Whereas most RNP enzymes are stable in composition and harbor preformed active sites, the spliceosome, which removes noncoding introns from precursor messenger RNAs (pre-mRNAs), follows fundamentally different strategies. In order to provide both accuracy to(More)
  • Nicholas J Watkins, Véronique Ségault, Bruno Charpentier, Stephanie Nottrott, Patrizia Fabrizio, Angela Bachi +4 others
  • 2000
The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be(More)
RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA(More)
Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of(More)
Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding(More)
The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-beta binding (IBB) domain and a COOH-terminal m3G-cap--binding region that bears no structural similarity to known(More)
How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. To address this question, we compared the intranuclear trafficking kinetics of small nuclear ribonucleoprotein particles (snRNP) and non-snRNP proteins in the presence and absence of splicing activity.(More)
The conserved Prp19 (pre-RNA processing 19) complex is required for pre-mRNA splicing in eukaryotic nuclei. Recent RNAi screens indicated that knockdown of Prp19 complex subunits strongly delays cell proliferation. Here we show that knockdown of the smallest subunit, BCAS2/Spf27, destabilizes the entire complex and leads to specific mitotic defects in human(More)