Secondary structures for splice junctions in eukaryotic and viral messenger RNA precursors.

Thermodynamically stable secondary structures have been found for adjacent splice junctions of 17 introns from 10 eukaryotic and viral mRNA precursors. Complementary regions in these structures position the exons to be ligated in a favourable orientation for splicing. The possible role of these structures in a general splicing mechanism in described.     

Neural network detects errors in the assignment of mRNA splice sites.

The use of databanks in genetic research assumes reliability of the information they contain. Currently, error-detection in the manually or electronically entered data contained in the nucleotide sequence databanks at EMBL, Heidelberg and GenBank at Los Alamos is limited. We have used a subset of sequences from these databanks to train neural networks to recognize pre-mRNA splicing signals in human genes. During the training on 33 human genes from the EMBL databank seven genes appeared to disturb the learning process. Subsequent investigation revealed discrepancies from the original published papers, for three genes. In four genes, we found wrongly assigned splicing frames of introns. We believe this to be a reflection of the fact that splicing frames cannot always be unambiguously assigned on the basis of experimental data. Thus incorrect assignment appear both due to mere typographical misprints as well as erroneous interpretation of experiments. Training on 241 human sequences from GenBank revealed nine new errors. We propose that such errors could be detected by computer algorithms designed to check the consistency of data prior to their incorporation in databanks.     

A large inverted repeat sequence overlaps two acceptor splice sites in adenovirus

The distribution of nucleotide sequences resembling functional sites for mRNA splicing was examined by computer-directed searches in order to determine what factors may influence splice site selection in nuclear precursors. In particular, the distribution of large potentially stable hairpin structures or regions of extensive dyad symmetry was studied in adenovirus sequences. One region, spanning 106 nucleotides, was found at 66.4 map units, overlapping back-to-back acceptor sites for two mRNA molecules, those coding for the 100K protein and the 72K DNA binding protein, which are transcribed from opposite strands. This region displays exceptional dyad symmetry and is potentially capable of forming a single, highly stable hairpin when transcribed. It seems likely that the secondary structure as well as the primary structure of RNA plays a role in determining the correct splicing of these mRNA molecules.     

Secondary structure analysis of adenovirus tripartite leader

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.     

Recognition of 3' -processing sites of human mRNA precursors

We have developed a computer program POLYAH and an algorithmfor the identification of 3'-processing sites of human mRNAprecursors. The algorithm is based on a linear discriminantfunction (LDF) trained to discriminate real poly(A) signal regionsfrom the other regions of human genes possessing the AATAAAsequence which is most likely nonfunctional. As the parametersof LDF, various significant contextual characteristics of sequencessurrounding AATAAA signals were used. An accuracy of methodhas been estimated on a set of 131 poly(A) regions and 1466regions of human genes having the AATAAA sequence. When thethreshold was set to predict 86% of poly(A) regions correctly,specificity of 51% and correlation coefficient of 0.62 had beenachieved. The precision of this approach is better than forthe other methods and has been tested on a larger data set.POLYAH can be used through World Wide Web (at Gene-Finder Homepage: URL http: //dot.imgen.bcm.tmc.edu: 9331/gene-finder/ gf.html)or by sending files with uncharacterized human sequences tothe University of Houston or Weizmann Institute of Science e-mailservers.     

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties

A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10–17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34269, calculated pI 4.7), and a 235 nucleotide 3-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-termininal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.     

Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.     

Secondary structure is required for 3' splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.     

S1 sensitive sites in adenovirus DNA.

S1 nuclease has been used as a probe for regions of DNA secondary structure in supercoiled recombinant plasmids containing adenovirus (Ad) DNA sequences. In the sequences examined two S1 sensitive sites were identified in the left-terminal 16.5% of Ad 12 DNA, one of which aligned approximately with an inverted repeat region in the DNA sequence. In addition an S1 sensitive site was dictated by a potential cruciform structure in the region of the Ad 2 major late promoter. In contrast to the expected cleavage site at the loop of the cruciform, cleavage occurred at the base of the stem in the region of the TATA box. All three S1 sensitive sites identified were more sensitive to S1 than the endogenous sites in the parent plasmids.