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Udgivelsesdato: May

Promoters and enhancers are cis-acting elements that control gene transcription via complex networks of protein-DNA and proteinprotein interactions. Whereas promoters deal with putting in place the RNA polymerase, both enhancers and promoters can control transcriptional initiation and elongation. We have previously shown that promoter structure modulates alternative splicing, strengthening the concept of a physical and functional coupling between transcription and splicing. Here we report that the promoter effect is due to the control of RNA pol II elongation. We found that the simian virus 40 (SV40) transcriptional enhancer, inserted in fibronectin (FN) minigene constructs transfected into mammalian cells, controls alternative splicing by inhibiting inclusion of the FN extra domain I (EDI) exon into mature mRNA. Deletion analysis of enhancer subdomains and competitions in vivo with excess of specific enhancer DNA subfragments demonstrate that the "minimal" enhancer, consisting of two 72-bp repeats, is responsible for the splicing effect. The 72-bp repeat region has been reported to promote RNA pol II elongation. When transcription is driven by the α-globin promoter linked to the SV40 enhancer, basal EDI inclusion and activation by the SR (Ser-Arg-rich) protein SF2/ASF are much lower than with other promoters. Deletion of only one of the two 72-bp repeats not only provokes higher EDI inclusion levels but allows responsiveness to SF2/ASF. These effects are the consequence of a decrease in RNA pol II elongation evidenced both by an increase in the proportions of shorter proximal over full length transcripts and by higher pol II densities upstream of the alternative exon detected by chromatin immunoprecipitation. Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fededa, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.

International audience; rp5l B pre-mRNA, like many Saccharomyces cerevisiae primary transcripts, contains a secondary structure within its intron sequence. The structure is required for optimal in vivo splicing efficiency and includes two complementary regions near the 5' splice site and the branchpoint (UB1 and DB1, respectively). An intron-containing RNA was probed in vitro with RNase T1 and dimethyl sulfate (DMS), and is folded as expected. We have also examined in vitro splicing of rp5l B pre-mRNA, by analyzing the formation of splicing complexes as well as splicing products. Similar analyses were done with mutant pre-mRNAs containing shortened or lengthened UB1/DB1 base pairing regions. Our experiments indicate that the secondary structure acts at an early step of spliceosome assembly to aid the formation of U1 snRNP-containing commitment complexes. pre-mRNAs were probed with DMS in vivo and the folding takes place inside cells. The effect of the different UB1/DB1 interactions on in vivo splicing efficiency was also analyzed. The results are consistent with the idea that the intramolecular interaction takes place prior to or at the beginning of spliceosome assembly.

This work introduces the first use of fluorescent in situ hybridization (FISH) to detect the distribution of specific transcripts in Saccharomyces cerevisiae. We have applied this technique to analysis of reporter transcripts from a single, integrated copy, or multicopy plasmids. We have evaluated the effect of splice site deletions or the presence or absence of a terminator/cleavage site and demonstrated that both splicing and polyadenylation affect the export of these transcripts from the nucleus to the cytoplasm. Moreover, we show that the exported pre-mRNAs are substrates for nonsense codon-mediated decay through the UPF1 pathway. The work presented here demonstrates that the spatial distribution of transcripts will also be an important component of yeast RNA metabolism.