Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)

Approaches to link RNA secondary structures with splicing regulation

In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitating or by hindering the interaction with factors and small nuclear ribonucleoproteins (snRNPs) that regulate splicing. Moreover, the secondary structure could play a fundamental role in the splicing of yeast species, which lack many of the regulatory splicing factors present in metazoans. This review describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3-prime splice site.Comment: 21 pages, 7 figure

GC content around splice sites affects splicing through pre-mRNA secondary structures

<p>Abstract</p> <p>Background</p> <p>Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (<it>Homo sapiens</it>), mice (<it>Mus musculus</it>), fruit flies (<it>Drosophila melanogaster</it>), and nematodes (<it>Caenorhabditis elegans</it>) to further investigate this phenomenon.</p> <p>Results</p> <p>We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures.</p> <p>Conclusion</p> <p>All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.</p

Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability

BACKGROUND: The emergence of eukaryotes was characterized by the expansion and diversification of several ancient RNA-binding domains and the apparent de novo innovation of new RNA-binding domains. The identification of these RNA-binding domains may throw light on the emergence of eukaryote-specific systems of RNA metabolism. RESULTS: Using sensitive sequence profile searches, homology-based fold recognition and sequence-structure superpositions, we identified novel, divergent versions of the Sm domain in the Scd6p family of proteins. This family of Sm-related domains shares certain features of conventional Sm domains, which are required for binding RNA, in addition to possessing some unique conserved features. We also show that these proteins contain a second previously uncharacterized C-terminal domain, termed the FDF domain (after a conserved sequence motif in this domain). The FDF domain is also found in the fungal Dcp3p-like and the animal FLJ22128-like proteins, where it fused to a C-terminal domain of the YjeF-N domain family. In addition to the FDF domains, the FLJ22128-like proteins contain yet another divergent version of the Sm domain at their extreme N-terminus. We show that the YjeF-N domains represent a novel version of the Rossmann fold that has acquired a set of catalytic residues and structural features that distinguish them from the conventional dehydrogenases. CONCLUSIONS: Several lines of contextual information suggest that the Scd6p family and the Dcp3p-like proteins are conserved components of the eukaryotic RNA metabolism system. We propose that the novel domains reported here, namely the divergent versions of the Sm domain and the FDF domain may mediate specific RNA-protein and protein-protein interactions in cytoplasmic ribonucleoprotein complexes. More specifically, the protein complexes containing Sm-like domains of the Scd6p family are predicted to regulate the stability of mRNA encoding proteins involved in cell cycle progression and vesicular assembly. The Dcp3p and FLJ22128 proteins may localize to the cytoplasmic processing bodies and possibly catalyze a specific processing step in the decapping pathway. The explosive diversification of Sm domains appears to have played a role in the emergence of several uniquely eukaryotic ribonucleoprotein complexes, including those involved in decapping and mRNA stability

Deciphering the universe of RNA structures and trans RNA-RNA interactions of transcriptomes in vivo: from experimental protocols to computational analyses

The last few years have seen an explosion of experimental and computational methods for investigating RNA structures of entire transcriptomes in vivo. Very recent experimental protocols now also allow trans RNA–RNA interactions to be probed in a transcriptome-wide manner. All of the experimental strategies require comprehensive computational pipelines for analysing the raw data and converting it back into actual RNA structure features or trans RNA–RNA interactions. The overall performance of these methods thus strongly depends on the experimental and the computational protocols employed. In order to get the best out of both worlds, both aspects need to be optimised simultaneously. This review introduced the methods and proposes ideas how they could be further improved

The life span determinant p66Shc localizes to mitochondria where it associates with mtHsp70 and regulates trans-membrane potential.

none18ORSINI F.; MIGLIACCIO E.; MORONI M.; CONTURSI C.; RAKER V.A.; PICCINI D.; MARTIN-PADURA I.; PELLICCIA G.; TRINEI M.; BONO M.; PURI C.; TACCHETTI C.; FERRINI M.; MANNUCCI R.; NICOLETTI I.; LANFRANCONE L.; GIORGIO M.; PELICCI P.Orsini, F.; Migliaccio, E.; Moroni, M.; Contursi, C.; Raker, V. A.; Piccini, D.; MARTIN PADURA, I.; Pelliccia, G.; Trinei, M.; Bono, M.; Puri, C.; Tacchetti, Carlo; Ferrini, M.; Mannucci, R.; Nicoletti, I.; Lanfrancone, L.; Giorgio, M.; Pelicci, P