Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.     

Effect of intron size on splicing efficiency in retroviral transcripts

To study the effect of intron size on splicing efficiency we have varied the size of the avian leukosis virus (ALV) env mRNA intron in a cloned ALV genome. This was accomplished by deletion of ALV sequences or insertion of phage lambda DNA. The effect of these modifications on splicing was analyzed by microinjection of the modified clones into RSV(-) chicken cells. Viral env mRNA when transcribed and properly spliced within these cells complemented the RSV(-) env deficiency leading to the production of focus forming units. Using this assay it was shown that deletion of up to 3.7 kb of the 4.68 kb env intron did not inhibit correct splicing nor did insertion of up to 8 kb of phage lambda DNA prevent splicing. Our results indicate that intron size can be varied over a wide range without preventing splicing.     

Intron mutations that affect the splicing efficiency of the CYH2 gene of Saccharomyces cerevisiae

Summary To define the extent of intervening sequences required for efficient splicing of the CYH2 gene in Saccharomyces cerevisiae, we have constructed a series of intron mutations. Artificial intron extensions of more than 300 bp of the natural intron lead to an inhibition of splicing where-as intron deletions lead to a drastic improvement of the splicing efficiency. It is shown that deletion of a 32 bp sequence element within the intron is responsible for this drastic improvement.     

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.     

Listening to silence and understanding nonsense: exonic mutations that affect splicing

Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.     

RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.     

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.     

Characterization of the cis-acting contributions to avian hepadnavirus RNA encapsidation

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.     

Effect of 5'' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.     

Potential mechanisms of mutations that affect neuronal migration in man and mouse

Mutations in the genes that encode filamin-1, Lis1 and doublecortin are responsible for X-linked lissencephaly in man, whereas mutations in the genes that encode Cdk5, its activator p35 and the reelin-signaling pathway disturb migration and architectonic development in mice. To understand the action of genes that control neuronal migration and the phenotype of corresponding defects, it might be as important to consider the positioning of the nucleus as it is to consider the guidance of the leading process.     

Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis

The intron of the 23S rRNA gene of D. mobilis is excised from the pre-23S RNA at specific sites in vivo and subsequently ligated to form a stable circular RNA, with a normal 5'-3' phosphodiester bond, containing the entire intron sequence; 95% of this RNA codes for a protein of 194 amino acids that can be expressed in E. coli. Crude cell extracts from D. mobilis also induce a two-step slicing reaction in vitro, producing the same circular intron RNA but a low yield of ligated exons. Cleavage depends on the RNA structure adjacent to the cleavage site and yields a 3'-terminal phosphate. Splicing is enhanced by GTP, but does not require divalent metal ions. The cleavage and exon-splicing reactions resemble those found for tRNA introns in eukaryotes and a possible structural rationale for this similarity is considered together with its possible implications for the origin of eukaryotic rRNA and tRNA introns.     

Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA structure and the mechanisms of alternative splicing

Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structures play an under appreciated role in the regulation of alternative splicing. This review provides an overview of the basic aspects of splicing regulation and highlights recent progress in understanding the role of RNA secondary structure in this process.