Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices

Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY). Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method ▿ §

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, Bact, and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, Bact, and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing

Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

<div><p>Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.</p></div

Molecular Architecture of the Human Prp19/CDC5L Complex▿ †

Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of ∼20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome

Site-specific UV crosslinking of U2 SF3a/b and RES complex proteins to the intron region around the branch-site in the yeast spliceosomal B^{act ΔPrp2} complex.

<p>(A). Schematic representation of site-specifically labeled pre-mRNAs carrying a single ³²P-labeled phosphate 5’ of the guanosines shown in green. The RNA fragments remaining after digestion with RNase T1 are indicated by a box below the sequence. Spliceosomes were assembled on site-specifically labeled pre-mRNAs in splicing extracts of a yeast <i>prp2-1</i> strain carrying proteins tagged with the TAP-tag at their C termini. B^{act ΔPrp2} complexes were purified according to protocol 2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.s011" target="_blank">S1 Text</a>). (B). Purified B^{act ΔPrp2} complexes containing TAP-tagged U2 SF3a/b proteins were UV irradiated (+ lanes) or non-irradiated (–lanes). All samples were then digested with RNase T1 and subjected to immunoprecipitation with IgG Sepharose. Immunoprecipitates were analyzed by SDS-PAGE and subsequent western blotting with peroxidase anti-peroxidase (PAP) complex antibody (upper panel). The western blot shows bands of the expected size of the U2 proteins indicated (note that the TAP-tag increases the size of a protein by ca. 21 kDa). The autoradiography of the membrane is shown in the lower panel. (C) As in (B), except that purified B^actΔPrp2 complexes containing TAP-tagged RES complex proteins were used. The arrows indicate ³²P-labeled RNA fragments crosslinked to the respective proteins.</p

Dynamics of protein–pre-mRNA interactions around the branch-site.

<p>Schematic representation of site-specifically labeled pre-mRNAs as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.g002" target="_blank">Fig 2</a>. B^actΔPrp2 complexes were purified according to protocol 2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.s011" target="_blank">S1 Text</a>). Purified B^actΔPrp2 and reconstituted B* and C complexes were UV irradiated, digested with RNase T1, and analyzed by SDS-PAGE electrophoresis. The amounts and molarity of the eluted spliceosomes were calculated on the basis of the specific activity of the pre-mRNA and equal molar quantity of B^act, B* and C complexes were loaded onto the gel.The autoradiography of the gel is shown. Question marks indicate uncharacterized crosslinked proteins. The arrowheads point to crosslinked Prp8 and the dot indicates crosslinked Prp45. (B) Lighter exposure of the bottom half of the gel shown in panel (A). (C) Autoradiography of the bottom part of 2D gels comprising total proteins of purified, UV-irradiated and RNase-digested B^act, B* and C complexes, respectively, which show the crosslinking intensities of Pml1, Hsh49 and Snu17 in each of the complexes. (D) As in C, only that the upper part of 2D gels is shown. The asterisk indicates a contaminant crosslinked protein.</p