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  • RNA splicing occurs in spliceosomes which is

    2018-10-24

    RNA splicing occurs in spliceosomes, which is a complex consisting of RNA and proteins, including five small nuclear RNAs (snRNAs) (U1, U2, U4, U5, and U6 snRNA), hundreds of auxiliary protein factors, and small nuclear ribonucleoproteins (snRNPs) (Reed et al., 1988). The RNA splicing process is catalyzed by the stepwise interaction of snRNPs and other splicing factors, leading to the subsequent formation of spliceosome complexes, termed the E, A, B, Bact, and C complexes. Transesterification reactions that excise introns and splicing exons occur via two catalytic activations between the Bact and C complexes. To form a catalytically active spliceosome, the SART1-pre-assembled tri-snRNP U4-U6/U5 is recruited to the A complex after the splicing factor SF3b1 interacts with U2 auxiliary factor (U2AF), thereby forming the pre-catalytic spliceosome (Gozani et al., 1998; Makarova et al., 2001). The phosphorylation of SF3b1 produces the catalytically competent spliceosome from which the U1 and U4 snRNPs are released (Roybal and Jurica, 2010). These catalytically active complexes execute cleavage at the 5′ splice site and lariat formation, followed by 3′ splice site cleavage and exon ligation. When an intron is excluded from the pre-mRNA molecule, the exon-exon junction complex (EJC) is deposited upstream of the resulting exon-exon junctions for nuclear export of spliced mRNAs (Le Hir et al., 2000). The spliceosome is then disassembled, thereby allowing the released spliceosome components to be reused for additional rounds of splicing.
    Materials and methods
    Results
    Discussion In almost all hPSs, spliceosomes were immunostained in a speckled pattern in the nucleus, whereas most of the human somatic Calcitriol had weak or no signals. A nuclear extract of mitogen-stimulated proliferative peripheral blood lymphocytes was used as the antigen for the anti-spliceosome antibody (Clevenger and Epstein, 1984), which we used in this study. The spliceosomes from highly proliferating human cancer cells were clearly recognized by the anti-spliceosome antibody, giving a speckled pattern (Clevenger et al., 1985; Clevenger and Epstein, 1984; Clevenger et al., 1987a, 1987b). Although there are few reports of this anti-spliceosome antibody, previous observations and the data presented in this study indicated that the anti-spliceosome antibody specifically recognizes spliceosomes, dependent upon the cell type and cell state in highly proliferative and transcriptionally active cells, such as hPSs. Morphological and molecular changes, such as loss of somatic cell signature, gain of epithelial signature, and chromatin remodeling, are significantly induced during the early period of the reprogramming process to generate hiPSs (Apostolou and Hochedlinger, 2013; Mikkelsen et al., 2008; Stadtfeld et al., 2008). These changes might be accompanied by changes in transcriptional activity and post-transcriptional modification. We demonstrated that the appearance of the speckled spliceosome pattern is an important feature of cellular reprogramming. Through gene expression profile analysis during the reprogramming process, we detected that expression changes in splicing factors occurred in 71 genes, of which 21 genes were significantly up-regulated during the early reprogramming stage. Among these 21 genes, SNRAP1, SNRPD1, and PNN were identified as putative targets of pluripotency control, and expression changes in these genes substantially affected pluripotency by inhibiting hPS spliceosome assembly. Of note, down-regulation of SNRAP1, SNRPD1, or PNN suppressed the protein expression of SNRPD1, while down-regulation of either SNRAP1 or SNRPD1, but not PNN, suppressed the protein expression of phospho-SF3b1. Co-immunoprecipitation studies further confirmed physical interactions between SNRPA1, SNRPD1, CDC5L, and phospho-SF3b1. Similarly, CDC5L and PRPF18 are reportedly bound tightly to spliceosomes during the second step of transesterification and can be used to identify the C complex (Ajuh et al., 2000; Horowitz and Krainer, 1997; Jurica et al., 2002). Our results emphasized that SNRPD1 is functionally a novel modulator of hPS spliceosomal activation. However, it is still difficult to definitively conclude whether hPS spliceosomes are catalytically active/activated spliceosomes of complexes Bact or C.