5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells

This laboratory previously reported that 5-fluorouracil (FUra) increases dihydrofolate reductase (DHFR) precursor mRNA (pre-mRNA) levels relative to DHFR mRNA levels in a methotrexate-resistant KB cell line; these data suggested that incorporation of FUra into RNA may, in part, lead to cell death through the inhibition of mRNA processing (Will, C. L., and Dolnick, B.J. (1987) J. Biol. Chem. 262, 5433-5436). Utilizing a methotrexate-resistant KB cell line designated 1BT, we now report the kinetic basis for altered levels of DHFR RNA observed in FUra-treated cells. Long-term exposure to FUra had no effect on the steady-state level of DHFR pre-mRNA containing intron V or I. However, steady-state levels of total DHFR mRNA decreased 2.0-fold on a per cell basis in cells exposed to 1.0 microM FUra. No significant change in the half-life of total DHFR mRNA or pre-mRNA was observed in cells exposed to FUra (t1/2 = approximately 11.5 h and 50 min, respectively). Nuclear/cytoplasmic RNA labeling experiments demonstrated that the rate of nuclear DHFR RNA conversion to cytoplasmic DHFR mRNA decreased approximately 1.8-fold in FUra-treated cells. These results provide further evidence the FUra may inhibit processing of mRNA precursors and/or affect the stability of nuclear DHFR mRNA.     

A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.     

A premature termination codon in either exon of minute virus of mice P4 promoter-generated pre-mRNA can inhibit nuclear splicing of the intervening intron in an open reading frame-dependent manner.

How premature translation termination codons (PTCs) mediate effects on nuclear RNA processing is unclear. Here we show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice caused a decrease in the accumulated levels of doubly spliced R2 relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of the large intron splice sites and by mutation of the AUG that initiated translation of R1 and R2. In contrast to the PTC at nt 385, the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor the normal termination codon for NS2; however, it could be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 open reading frame. This suggested that there was recognition and communication of reading frame between exons on a pre-mRNA in the nucleus prior to or concomitant with splicing.     

Evolutionary divergence of exon flanks: a dissection of mutability and selection

The intronic sequences flanking exon-intron junctions (i.e., exon flanks) are important for splice site recognition and pre-mRNA splicing. Recent studies show a higher degree of sequence conservation at flanks of alternative exons, compared to flanks of constitutive exons. In this article we performed a detailed analysis on the evolutionary divergence of exon flanks between human and chimpanzee, aiming to dissect the impact of mutability and selection on their evolution. Inside exon flanks, sites that might reside in ancestral CpG dinucleotides evolved significantly faster than sites outside of ancestral CpG dinucleotides. This result reflects a systematic variation of mutation rates (mutability) at exon flanks, depending on the local CpG contexts. Remarkably, we observed a significant reduction of the nucleotide substitution rate in flanks of alternatively spliced exons, independent of the site-by-site variation in mutability due to different CpG contexts. Our data provide concrete evidence for increased purifying selection at exon flanks associated with regulation of alternative splicing.     

Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA.

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations within the TPI gene that cause translation to terminate prematurely at or upstream of codon 189, within exon 6, result in a decreased level of TPI mRNA (I.O. Daar and L.E. Maquat, Mol. Cell. Biol. 8:802-813, 1988). For all mutations in this group, the decrease is to the same extent, i.e., to approximately 20% of the normal level. We show here that a second group of nonsense mutations that cause translation to terminate prematurely at or downstream of codon 208, in exon 6, did not affect TPI mRNA abundance. Deletion analysis demonstrated that the abundance of translationally active TPI mRNA is a function of both the distance and the polarity of the nonsense codon relative to the final intron in TPI pre-mRNA. Our results indicate that if translating ribosomes are unable to progress to at least a certain position within the penultimate exon relative to the final intron, then the level of the corresponding mRNA will be abnormally low. Studies inhibiting RNA synthesis with dactinomycin demonstrated that a block in translation does not affect the half-life of mature TPI mRNA. The simplest interpretation of our data is that the translation of TPI mRNA in the cytoplasm facilitates the splicing of TPI pre-mRNA or the transport of TPI mRNA across the nuclear envelope or both.     

Association of nuclear matrix antigens with exon-containing splicing complexes

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser- Arg family as a component of the nuclear matrix.     

Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types. To study CT specific processing in Hela cell nuclear extracts we have used precursor RNAs corresponding to the exon 3 to exon 4 region containing only CT specific processing signals. The results revealed the usage of a uridine residue 23 nucleotides upstream of the 3' splice site as the major site of lariat formation in CT specific splicing. The implications of this finding for the alternative, tissue specific processing of the CALC-I pre-mRNA and for branch point selection in general are discussed.     

The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.     

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.