Heat shock affects 5′ splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles

Abstract

The effect of heat shock on the packaging and splicing of nuclear CAD pre-mRNA, a transcript expressed constitutively from a non heat-inducible promoter, was studied in vivo in Syrian hamster cells. While mild heat shock did not affect significantly the packaging of CAD RNA in 200S InRNP particles, it caused perturbation to splicing. First, the heat shock inhibited splicing of CAD pre-mRNA. Second, it affected 5' splice site selection by activating cleavage at a cryptic 5' splice site; yet ligation of the cryptic exon to the downstream proximal exon was not observed. Base complementarities of the cryptic site with U1, U5, or U6 snRNAs are comparable, or even better, than those with the neighboring normal site. Hence, the exclusion of the cryptic site under normal growth conditions cannot be attributed to weaker base pairing with these snRNAs. On the other hand, these results imply the involvement of a heat labile factor in the selection of the 5' cleavage site. The exclusion of the cryptic site at 37 degrees C and the aborted splicing at this site after heat shock may also be explained by a proposed nuclear checking mechanism that detects in-frame stop codons upstream of the 5' splice site, and aborts splicing at such sites to prevent the production of a defective message.

Stop codons affect 5′ splice site selection by surveillance of splicing

“…A clue for a possible mechanism arises from the fact that the intron sequences upstream to 94.5% of the intronic latent sites contain at least one stop codon in the reading frame determined by the bona fide upstream exons. This general finding conforms with the idea that the cell's nucleus harbors a checking mechanism that is capable of recognizing premature stop codons in premRNAs, and consequently aborting splicing at downstream latent 5Ј SSs (5).…”

“…Syrian hamster SV28 fibroblast cells and human 293T cells were grown to 50% confluence in tissue culture plates as described (5) and transiently transfected with the appropriate DNA constructs (10 g or 2 g per 5 ϫ 10 6 SV28 or 293T cells, respectively) by using the calcium phosphate method (10). Cells were harvested 24 or 48 h after transfection, and total cellular RNA was extracted with guanidinium thiocyanate as described (5). Treatment with antibiotics was as described in Fig.…”

“…Nonetheless, its involvement in splicing never occurred under normal growth conditions. It could, however, be activated for splicing under stress conditions (5), indicating its capability to serve as a splice donor. The presence of in-frame stop codons upstream of the latent SS raised the possibility that splicing at this latent site under normal growth conditions was aborted, thus eliminating the formation of an mRNA that contains premature translation termination codons (PTCs).…”

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

“…The full-length 150-kDa form of ADAR1 was found in a broad peak around the 200S region of the gradient (Fig. 1A Upper), where specific nuclear pre-mRNAs have been shown to sediment (19,(38)(39)(40). The more abundant shorter (110 kDa) form of ADAR1 had a broader distribution, with significant level sedimenting at the 200S and 70S regions of the gradient ( Fig.…”

Regulation of splicing: The importance of being translatable: FIGURE 1.

“…Latent sites appear to be highly abundant in the genome, particularly in introns of protein-coding genes (see below). To explain the phenomenon of suppression of splicing (SOS) at latent 5Ј splice sites, we proposed that the necessity to maintain the translatability of mRNAs, by avoiding the inclusion of premature termination codons in them, could serve as a criterion that differentiates normal 5Ј splice sites from latent ones (Miriami et al 1994). We substantiated this proposal by showing, in two gene systems, that an intronic latent 5Ј splice site can be activated if all upstream stop codons, which are in the reading frame of the upstream exon, are eliminated by point or frame-shift mutations .…”