The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing

Sedimentation profiles of U1-70K (Usp101p), U1H (Usp107p), U1J (Usp108) and U1L (Usp109p) complexes

<p><b>Copyright information:</b></p><p>Taken from "Proteomic analysis of the U1 snRNP of reveals three essential organism-specific proteins"</p><p></p><p>Nucleic Acids Research 2007;35(5):1391-1401.</p><p>Published online 30 Jan 2007</p><p>PMCID:PMC1865046.</p><p>© 2007 The Author(s).</p> () Protein extract (5 mg) of cells expressing U1-70K-TAP, U1H-TAP and U170-HA or U1J-TAP and U1-70K-HA or U1L-TAP and U1-10K-HA were separated on a 10–30% glycerol gradient. The gradient fractions () were precipitated with IgG antibodies and separated by SDS-PAGE, immunoblotted and probed with αTAP antibodies to determine the distribution of U1-70K-TAP, U1H-TAP, U1J-TAP and U1L-TAP, as indicated. () The gradient fractions 3–8 and 10–15 of a gradient-containing U1L-TAP and U1-70K-HA were pooled bound to IgG-Sepharose. The bound material was separated on SDS-PAGE, immunoblotted and probed with IgG and HA antibodies. An aliquot (50%) of the bound material was used to isolate RNA and hybridized with a labeled U1 probe to visualize U1 snRNA as indicated. Mo, pooled gradient fractions 10–15 of a protein extract from a wild-type strain. The pooled gradient fractions 3–8 from this gradient also showed no signals (not shown). The gradient was calibrated with small (30S) and large (50S) ribosomal subunits from