Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

Abstract

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) In view of the proposed role of the SR proteins in premRNA splicing, this family of proteins is expected to be associated with the RNA processing machinery in vivo. PremRNA splicing in vivo apparently occurs on structures more complex than individual spliceosomes. This should not be surprising, because the splicing machinery of a living cell is expected to package and process multi-intronic pre-mRNAs, rather than the mono-intronic pre-mRNAs, which are typically used to assemble spliceosomes in vitro. Indeed, we have shown that several specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A) + RNA, are packaged in compact large nuclear RNP (lnRNP) particles that invariably sediment at the 200S region in sucrose gradients (31)(32)(33)(34) 8830The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

“…We previously have shown that when HeLa cell nuclear supernatants were fractionated in sucrose gradients and probed for a specific pre-mRNA (e.g., ␤-actin), a relatively broad but distinct peak of the specific RNA was observed at the 200S region of the gradient (38). All of the nucleoplasmic phosphorylated SR proteins also sedimented as one peak at 200S and were associated exclusively with 200S lnRNP particles (22). Other components of the lnRNP particles, such as the U snRNPs, U2AF, and PTB (19,22) gave a broader distribution across the sucrose gradients and were particularly abundant in smaller complexes that sedimented closer to the top of the gradient.…”

“…These processes have been proposed to be carried out within large nuclear ribonucleoprotein (lnRNP) particles that sediment at 200S in sucrose gradients. The composition of the lnRNP particles (19)(20)(21)(22) is very similar to that of the in vitro-assembled spliceosome (reviewed in refs. 23 and 24).…”

“…23 and 24). Specifically, all of the U small nuclear ribonucleoproteins (snRNPs) required for pre-mRNA splicing, as well as several non-snRNP protein splicing factors, including U2AF and the SR protein family (23)(24)(25) are present within the lnRNP particles (19,21,22).…”

The editing enzyme ADAR1 and the mRNA surveillance protein hUpf1 interact in the cell nucleus

“…Nuclear pre-mRNAs together with all pre-mRNA processing components are packaged in supraspliceosomes that represent the native pre-mRNA processing machine (26)(27)(28)(29). These complexes contain all five spliceosomal U small nuclear ribonucleoproteins (snRNPs) (25), as well as splicing factors such as the SR protein family (30), and the ADAR A-to-I RNA editing enzymes (26). Because hUpf1 protein was shown to be involved in NAS (20), we reasoned that it might be associated with supraspliceosomes.…”

“…Indirect IPs were performed as described in ref. 30 by using anti-ADAR1 WI9 antibodies (kindly provided by K. Nishikura), antihUpf1 antibodies, or anti-Sm mAb Y12 or a nonrelevant antibody, preimmune anti-rabbit IgG (see SI Materials and Methods). IP yield is expressed as the ratio between the intensity of the immunoprecipitated band [minus background of control lanes (lanes 2 and 3)] and the sum of this intensity and that of the corresponding band in the unbound lane.…”