No abstract

We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5 untranslated regions (5 untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.[Keywords: splicing; pre-mRNA; motifs; exon; enhancers; silencers] Supplemental material is available at http://www.genesdev.org.

Mutants of Chinese hamster ovar cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to hi -pcificactivity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3Hjdeoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heatlabile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene. (6); (fi) DHFR that is intrinsically less sensitive to MTX (6, 7); or (iii) overproduction of DHFR activity (6,8,9). The last class appears to be the most common, and the overproduction has been shown to result from increased synthesis of wild-type enzyme (10, 11) due to dhfr gene amplification (12). (17) fetal calf serum whenever cell nutrition was being manipulated. Cells were grown at 370C in an atmosphere of 5% carbon dioxide.Selection of DHFR-Deficient Mutants. Mutagenesis with ethyl methanesulfonate (EtMes) and selection of 6-thioguanine-resistant mutants have been described (18). Mutagenesis with y rays was carried out by immersing vials containing cell suspensions in a water tank containing a cobalt-60 source for varying time intervals. The dose used for the isolation of mutants described in Table 2 was 690 rads (1 rad = 1.00 X 10-2 J/kg), which reduced viability to 9%.A partially DHFR-deficient, presumptive

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 39 splice sites tend to be single stranded, and effective 59 splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.[Supplemental material is available for this article.]The transfer of genetic information from DNA to protein in living things is accomplished with accuracy, precision, and fidelity. These qualities characterize pre-mRNA splicing as much as transcription and translation (Fox-Walsh and Hertel 2009). The accurate identification of splice sites in long metazoan transcripts depends not only on the splice-site sequences that are substrates for the splicing reaction, but also on short RNA stretches known as exonic and intronic splicing enhancers (ESEs and ISEs) and silencers (ESSs and ISSs). These so-called splicing regulatory motifs are manifold and,

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.In an effort to identify and understand those aspects of gene structure that play a role in gene expression in mammalian cells, we have been carrying out a detailed mutational analysis of the dihydrofolate reductase (dhfir) gene in Chinese hamster ovary (CHO) cells. dlifr exhibits many typical characteristics of mammalian housekeeping genes: it is 25 kilobase pairs (kbp) in size, contains five variously sized introns, and has a promoter region with no TATA box but with a very high G+C content and an Spl binding site (10,22,40,53). The basic strategy has been to isolate mutants that are deficient in DHFR enzyme activity; such mutants can be readily selected from a CHO cell line that is hemizygous at this locus (58, 59). To focus on lesions that may affect transcription or RNA processing, mutants can be screened for alterations that have affected dimfi-mRNA, either qualitatively or quantitatively.We have previously described spontaneous point mutations in th...