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

AUTOR(ES)

Woppmann, A

RESUMO

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'.

Documentos Relacionados

• The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.
• Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts.
• The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.
• The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.
• Direct cross-linking of snRNP proteins F and 70K to snRNAs by ultra-violet radiation in situ.