Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome. Currently, little is known about the specific roles of these factors in splicing. In this study, we characterized the molecular interaction between Hprp3p and Hprp4p. Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates. By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N-or Cterminal region did not interact with Hprp4p. In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts. Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.Pre-mRNA splicing occurs in the spliceosome, a large RNAprotein complex that contains a pre-mRNA, four essential small nuclear ribonucleoprotein (snRNP) 1 particles (U1, U2, U5, and U4/U6), and numerous non-snRNP splicing factors (1-3). Each snRNP particle consists of one (U1, U2, and U5) or two (U4/U6) snRNAs complexed with a set of Sm or Sm-like proteins and several particle-specific proteins (4 -6). These snRNPs recognize conserved sequences of the pre-mRNA and assemble into a catalytically active spliceosome that catalyzes the two cleavage-ligation reactions of pre-mRNA splicing (1,2,7,8). Spliceosome assembly follows an ordered pathway through the formation of several intermediate complexes (2). First, a pre-spliceosome (A complex) is formed once U1 and U2 snRNAs (as part of the U1 and U2 snRNPs) associate with the conserved 5Ј-splice site and the branch point of the intron, respectively (9, 10). Then, U4/U6 snRNP, in which U4 and U6 snRNAs base pair over an extended complementary region, forming a Y-shaped junction, is recruited together with U5 snRNP, presumably via protein/protein interactions, to the pre-spliceosome to form the mature spliceosome (B complex) (11-13).Prior to the first catalytic step of splicing, important conformational rearrangements occur to create a catalytically active spliceosome. For example, U1 snRNA dissociates from the 5Ј-splice site, and the U4/U6 snRNA association is disrupted (14 -16), leaving the U6 snRNA free to base pair with both the U2 snRNA and the 5Ј-splice site (17). The U2 and U6 snRNAs, together with the pre-mRNA, may form the catalytic core of the s...
Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.