Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a common mechanism of regulating gene expression. SR proteins are a family of pre-mRNA splicing factors that are structurally related and evolutionarily conserved. Any member of the SR family can complement a splicing-deficient extract that lacks the entire family of SR proteins. Here it is demonstrated that particular SR proteins have distinct functions in alternative pre-mRNA splicing in vitro. In addition, SR proteins are differentially expressed in a variety of tissues. These results suggest a fundamental role for SR proteins in the regulation of alternative splicing.

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3 ′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3 ′ ends.

mRNA is packaged into ribonucleoprotein particles called mRNPs. A multitude of RNA-binding proteins as well as a host of associated proteins participate in the fate of mRNA from transcription and processing in the nucleus to translation and decay in the cytoplasm. Methodological innovations in cell biology and genome-wide high-throughput approaches have revealed an unexpected diversity of mRNA-associated proteins and unforeseen interconnections between mRNA-processing steps. Recent insights into mRNP formation in vivo have also highlighted the importance of mRNP packaging, which can sort RNAs on the basis of their length and determine mRNA fate through alternative mRNP assembly, processing and export pathways.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

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