Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage

Recognition of 5' splice sites in pre-mRNA splicing is achieved in part by base pairing with U1 snRNA. We have used interactive suppression in the yeast Saccharomyces cerevisiae to look for other factors involved in 5' splice-site recognition. This approach identified an extragenic suppressor that activates a cryptic 5' splice site. The suppressor is a gene for U5 snRNA (snR7) with a single base mutation in a strictly conserved 9 base sequence. This suggests that U5 snRNA can play a part in determining the position of 5' splice-site cleavage. Consistent with this, we have been able to isolate other mutations in the 9 base element in U5 snRNA that specifically activate a second cryptic 5' splice site nearby.     

The identical 5' splice-site acceptor mutation in five attenuated APC families from Newfoundland demonstrates a founder effect

Inherited mutations of the APC gene predispose carriers to multiple adenomatous polyps of the colon and rectum and to colorectal cancer. Mutations located at the extreme 5' end of the APC gene, however, are associated with a less severe disease known as attenuated adenomatous polyposis coli (AAPC). Many individuals with AAPC develop relatively few colorectal polyps but are still at high risk for colorectal cancer. We report here the identification of a 5' APC germline mutation in five separately ascertained AAPC families from Newfoundland, Canada. This disease-causing mutation is a single basepair change (G to A) in the splice-acceptor region of APC intron 3 that creates a mutant RNA without exon 4 of APC. The observation of the same APC mutation in five families from the same geographic area demonstrates a founder effect. Furthermore, the identification of this germline mutation strengthens the correlation between the 5' location of an APC disease-causing mutation and the attenuated polyposis phenotype.     

Comparative analysis of sequences at the 5' end of the human and mouse apolipoprotein B genes

A comparison was made between the DNA sequences in two regions of the mouse and the human apolipoprotein B genes: the 5'-flanking sequence and the region between the first exon and the second intron. Considerable homology was observed, particularly in the immediate 5' region and in the second intron. Because promoter and enhancer elements have been previously localized to these regions in the human apolipoprotein B gene, it is proposed that regions of conserved base sequence delineate binding regions for regulatory proteins. In some cases, contiguous regions of homology are longer than expected for regions designed as recognition sites for individual nuclear proteins, and may define regions recognizable by a cluster of interacting proteins. Both the human and mouse genes contain repetitive elements and a hypervariable dinucleotide repeat.     

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

Human Ras converting enzyme endoproteolytic specificity at the P2' and P3' positions of K-Ras-derived peptides

The membrane associated endoprotease, hRCE1, is responsible for one step in Ras membrane localization. The "CAAX" sequence at the C-terminal of farnesylated Ras proteins is cleaved by hRCE1 to yield an AAX tri-peptide. We found that an 8-aa K-Ras-derived "CAA" peptide, KSKTKC(farnesyl)VI, was a better substrate for hRCE1 than a KSKTKC(f)VIM "CAAX" peptide. When we examined hRCE1 activity on the same K-Ras core peptide with H-Ras (VLS) or N-Ras (VVM) C-terminal AAX sequences, we also found that in each case, the CAA peptides were better hRCE1 substrates. For each peptide set we examined, the P2' (A) and P3' (X) positions appeared independent in influencing hRCE1 activity on peptide substrates. We found that at the P3' position, methionine was better than serine; while at the P2' position, isoleucine and valine were better than leucine. Additionally, we found that a similar noncleaved peptide (modified at P'2 with a nitrophenyl group) could act as a competitive inhibitor of the reaction. Thus, hRCE1 has important functional interaction with the P2' and P3' substrate positions in addition to the farnesylated cysteine at the scissile bond site. This data could be useful in design of peptidomimetic inhibitors of hRCE1. Such inhibitors may be useful in treatment of cancer and inflammatory disease.     

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.     

A fast method of analysis of the 5' terminal nucleotides of deoxyribooligonucleotides

A method for the rapid analysis of the 5′ ends of deoxyribooligonucleotides is described. The method involves: (a) labeling of the 5′ ends with polynucleotide kinase; (b) digestion of the oligonucleotides to 5′ mononucleotides with pancreatic DNAase and snake venom exonuclease; (c) unidimensional separation on polyethyleneimine plates of the four 5′ mononucleotides, ATP, and inorganic phosphate. This method requires only a minimum amount of work, time, and material.     

Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3' splice-site consensus sequence.

The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, characterizes the genes of nuclear encoded mitochondrial proteins.     

Functional analyses of positions across the 5' splice site of the trypanosomatid spliced leader RNA. Implications for base-pair interaction with U5 and U6 snRNAs

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced leader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K. P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8473-8478) and the interaction of the SL RNA intron with U6 snRNA analogous to cis-splicing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 relative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs. All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon positions for trans-splicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cis-splicing, but position +2 had the most severe effect on trans-splicing. Compensatory mutations were introduced in U5 and U6 snRNAs to examine whether the defects resulted from failure to interact with these snRNAs by base pairing. Suppression was observed only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failure to suppress the other SL RNA mutants by the U5 compensatory mutations suggests that another factor(s) interacts with these key SL RNA positions.