Structural basis for dual roles of Aar2p in U5 snRNP assembly

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p–RNase H platform. Known Brr2p interaction sites of the Jab1/MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phospho-mimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly

USP15 regulates dynamic protein-protein interactions of the spliceosome through deubiquitination of PRP31.

Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial-mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators

PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex

A unified mechanism for intron and exon definition and back-splicing.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes

Interaction of yeast eIF4G with spliceosome components Implications in pre-mRNA processing events

International audienceAs evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism