Huntriss, John D'Arcy; (1995) Clinical and functional investigations of the tissue specific SmN protein. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (19MB)

Abstract

The SmN protein is highly homologous to the constitutively expressed small nuclear ribonucleoprotein particle (snRNP)-associated protein SmB but is expressed in brain and heart only. SmN is demonstrated here to associate with snRNPs when expressed naturally or artificially and interestingly, the nature of this association is different to that exhibited by SmB. SnRNPs catalyse RNA splicing of the primary trancripts within the spliceosome to generate mRNAs encoding for proteins. The primary transcripts of some genes however, are processed differently according to the tissue or cell type to generate distinct mRNAs encoding for different proteins. The tissue specific expression of SmN suggests it plays an important RNA splicing role in the tissues expressing it, and hence is a putative regulator of alternative splicing. The gene encoding SmN in humans, SNRPN has been mapped to the critical region for Prader Willi Syndrome (PWS) in which patients lack a functional paternally inherited copy of the maternally imprinted SNRPN gene. Murine models of PWS that lack expression of the mouse homologue (Snrpn) suffer early postnatal lethality. Data presented here confirms the absence of SmN expression at the protein level in all tissues tested demonstrating that the maternal imprint is not relaxed in these mice. Furthermore, SmB expression was observed to be elevated in the brains of such mice. This suggests that elevated SmB expression cannot compensate for important splicing roles normally performed by SmN in the tissues expressing it. RNA from the brains of these mice was therefore used to study the proposed alternative splicing role of SmN. Genes proposed to be good candidates for regulation by SmN on the basis that their pattern of splicing is different in tissues expressing SmN were analysed by RT-PCR amplification of endogenous transcripts. Analysis of c-src, NCAM the Oct-2 transcription factor, CGRP and the GS alpha and Go alpha subunits of GTP binding proteins argues against a general role of SmN in alternative splicing, but the fate of these mice suggests SmN has unique functions or properties not assigned to SmB. This is consistent with a functional analysis by immunoprecipitation whereby SmN demonstrated different affinities for the U1 and U2 snRNPs, showing preferential association with the U2 snRNP at low levels of expression. In addition, ELISA screening using peptides corresponding to the regions of least sequence homology between SmN and SmB/B' revealed that a subset of Systemic Lupus Erythematosus (SLE) patients generate autoantibodies that can discriminate between and SmN and SmB/B' by binding an SmB/B'-specific epitope (SmB/B' 179- 190). Therefore the differences in primary structure between SmN and SmB/B' are sufficient to alter both protein conformation and the nature of association with snRNPs, implying that SmN- containing snRNPs may be capable of processing RNA differently. In view of these findings, further studies have been performed to determine whether a reported significant elevation in gene transcription of SNRPN in certain SLE patients was reflected at the protein level. Data presented in this study suggests a post-transcriptional control mechanism operates to counter this aberrant SNRPN transcription and that elevated SmN protein expression is not a common occurrence in SLE. This is consistent with both immunoprecipitation data and ELISA data in that SmN does not appear to be the antigen stimulating the immune system in SLE.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item