In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.Biogenesis of proteins in eukaryotes is a multistep process that involves the concerted action of several complex machineries. Multiprotein complexes containing RNA polymerase II are involved in transcribing genes into pre-messenger RNA. Most human genes contain introns that are removed by splicing, a process orchestrated and catalyzed by the large multiprotein/ RNA complex termed the spliceosome. Polyadenylation of the mRNA is also catalyzed by a complex processing machinery before mRNAs are exported to the cytosol, where translation by ribosomes takes place. Although much is known about the individual processes in protein biogenesis, how the separate steps are integrated is much less clear.The spliceosome is comprised of five small nuclear RNAs (snRNAs)-U1, U2, U4, U5, and U6 snRNA-as well as many protein factors (Staley and Guthrie 1998). Some of these proteins are tightly associated with the snRNAs, forming small nuclear ribonucleoproteins (snRNPs) that are thought to assemble in a stepwise manner onto the pre-mRNA to form the spliceosome. Work over the last decade has elucidated the temporal sequence of recognition of the splice sites by the respective snRNPs and protein factors (Hastings and Krainer 2001). Interestingly, the view of stepwise assembly of the spliceosome has recently been challenged in favor of a more concerted mechanism involving preformed spliceosomes (Stevens et al. 2002). Besides the snRNP subunits, a large number of non-snRNP proteins are known, which perform various functions during the splicing reaction. For example, multiple members of the DEAD-box helicase family are thought to control RNA base-pairing interactions at different stages of spliceosome assembly and catalysis, whereas members of the SR motif family are believed to be link factors promoting protein-protein interactions during spliceosome assembly. In all, ∼100 different proteins have been linked to splicing through biochemical and/or genetic evidence (for review, see Will and Lührmann 1997). However, it remains unclear how complete this list might be.In an alternative systematic approach to the traditional characterization of single splicing factors, the spliceosome can be purified and...

Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy‐dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS‐binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino‐terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1‐like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino‐terminal domain inhibits import of NLS‐containing proteins when added to an in vitro nuclear transport assay. While full‐length hSRP alpha is able to leave the nucleus, the amino‐terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS‐containing substrates to the protein import machinery.

2'-O-Methyloligoribonucleotides have been synthesised on solid phase from base protected 5'-O-dimethoxytrityl-2'-O-methylribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2'-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2'-O-methyloligoribonucleotide probes carrying several 5'- terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2'-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.

No abstract

No abstract