Sensitivity of splice sites to antisense oligonucleotides in vivo.

A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

An algorithm to predict 3 ' intron splice sites in Plasmodium falciparum genomic sequences

A new algorithm, PfAGSS, for predicting 3' splice sites in Plasmodium falciparum genomic sequences is described. Application of this program to the published P. falciparum chromosome 2 and 3 data suggests that existing programs result in a high error rate in assigning 3' intron boundaries. (C) 2001 Elsevier Science B.V. All rights reserved.

Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

Comparison of splice sites in mammals and chicken

We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by background intron conservation. In contrast, additional conservation over background is detectable in orthologous mammalian and chicken splice sites. Our results also indicate that the U2 and U12 intron classes seem to have evolved independently since the split of mammals and birds; we have not been able to find a convincing case of interconversion between these two classes in our collections of orthologous introns. Similarly, we have not found a single case of switching between AT-AC and GT-AG subtypes within U12 introns, suggesting that this event has been a rare occurrence in recent evolutionary times. Switching between GT-AG and the noncanonical GC-AG U2 subtypes, on the contrary, does not appear to be unusual; in particular, T to C mutations appear to be relatively well tolerated in GT-AG introns with very strong donor sites.

Assessment of transcript reconstruction methods for RNA-seq.

We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon–intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

SpliceDB: database of canonical and non-canonical mammalian splice sites

A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT–AG junctions (22 199 entries) and 0.56% have non-canonical GC–AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC–AG pairs (of which one was an error that corrected to GC–AG), 61 errors corrected to GT–AG canonical pairs, six AT–AC pairs (of which two were errors corrected to AT–AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: http://genomic.sanger.ac.uk/spldb/SpliceDB.html and at http://www.softberry.com/spldb/SpliceDB.html.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Differences in the RNA binding sites of iron regulatory proteins and potential target diversity.

Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is an important mechanism for the regulation of gene expression. The members of the SR protein family of pre-mRNA splicing factors have distinct functions in promoting alternative splice site usage. Here we show that SR proteins are required for the first step of spliceosome assembly, interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Further, we find that individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA.