Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants

Abstract

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

Protein components specifically associated with prespliceosome and spliceosome complexes.

“…On the basis of comparisons with published two-dimensional gels {Feeney et Woppmann et al 1990), however, we do detect lower molecular weight proteins that are likely to correspond to the known snRNP core proteins (C, D, E, F, and G; data not shown). Notably, other than these proteins, we do not detect significant levels of any other lower molecular weight proteins.…”

Sex-lethalsplicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping

“…Interestingly, of the two U1-70K isoforms observed in whole cell extracts, only the more rapidly migrating U1-70K species was identified in the pull-down experiments. As U1-70K is known to be phosphorylated (Tazi et al, 1993;Woppmann et al, 1990), we suspected that the more rapidly migrating form might be dephosphorylated. Consistent with this notion, we found that phosphatase treatment of the embryonic extracts prior to western blot analysis resulted in a single U1-70K species with a similar mobility to the protein detected in the pull down experiments (data not shown).…”

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

“…It is thought that the Ser residues in the RS domains of SR proteins become phosphorylated, since all are phosphoproteins (Krainer et al, 1991;Zahler et al, 1992) and the analogous domain in U1-70K is phosphorylated at multiple serines in vivo (Woppmann et al, 1990). Moreover, synthetic random co-polymers of Arg and Ser are excellent substrates for many Ser/Thr protein kinases (Racker, 1991).…”