Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly.
We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74 kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74 kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5' exon-intron part, rather than the intron-3' exon part, of pre-mRNA was observed.
Kafasla
Panayiota
P
Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 11635 Athens, Greece.
Patrinou-Georgoula
Meropi
M
Lewis
Joe D
JD
Guialis
Apostolia
A
D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D005810:Multigene Family; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D006163:Ribonuclease T1 / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013379:Substrate Specificity
D012322:RNA Precursors; D012261:Ribonucleoproteins; D006163:Ribonuclease T1