Branch point selection in alternative splicing of tropomyosin pre-mRNAs.

The rat tropomyosin 1 gene gives rise to two mRNAs encoding rat fibroblast TM-1 and skeletal muscle beta-tropomyosin via an alternative splicing mechanism. The gene is comprised of 11 exons. Exons 1 through 5 and exons 8 and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle whereas exons 7 and 10 are used exclusively in skeletal muscle. In the present studies we have focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). To study the mechanism and regulation of alternative splice site selection we have characterized the branch points used in processing of the tropomyosin pre-mRNAs in vitro using nuclear extracts obtained from HeLa cells. Splicing of exon 5 to exon 6 (fibroblast-type splice) involves the use of three branch points located 25, 29, and 36 nucleotides upstream of the 3' splice site of exon 6. Splicing of exon 6 (fibroblast-type splice) or exon 7 (skeletal muscle type-splice) to exon 8 involves the use of the same branch point located 24 nucleotides upstream of this shared 3' splice site. In contrast, the splicing of exon 5 to exon 7 (skeletal muscle-type splice) involves the use of three branch sites located 144, 147 and 153 nucleotides, upstream of the 3' splice site of exon 7. In addition, the pyrimidine content of the region between these unusual branch points and the 3' splice site of exon 7 was found to be greater than 80%. These studies raise the possibility that the use of branch points located a long distance from a 3' splice site may be an essential feature of some alternatively spliced exons. The possible significance of these unusual branch points as well as a role for the polypyrimidine stretch in intron 6 in splice site selection are discussed.     

Determinants of Nam8-dependent splicing of meiotic pre-mRNAs

Nam8, a component of yeast U1 snRNP, is optional for mitotic growth but required during meiosis, because Nam8 collaborates with Mer1 to promote splicing of essential meiotic mRNAs AMA1, MER2 and MER3. Here, we identify SPO22 and PCH2 as novel targets of Nam8-dependent meiotic splicing. Whereas SPO22 splicing is co-dependent on Mer1, PCH2 is not. The SPO22 intron has a non-consensus 5' splice site (5'SS) that dictates its Nam8/Mer1-dependence. SPO22 splicing relies on Mer1 recognition, via its KH domain, of an intronic enhancer 5'-AYACCCUY. Mutagenesis of KH and the enhancer highlights Arg214 and Gln243 and the CCC triplet as essential for Mer1 activity. The Nam8-dependent PCH2 pre-mRNA has a consensus 5'SS and lacks a Mer1 enhancer. For PCH2, a long 5' exon and a non-consensus intron branchpoint dictate Nam8-dependence. Our results implicate Nam8 in two distinct meiotic splicing regulons. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. The leader, tail and RRM1 are dispensable for splicing meiotic targets and unnecessary for vegetative Nam8 function in multiple synthetic lethal genetic backgrounds. Nam8 activity is enfeebled by alanine mutations in the putative RNA binding sites of the RRM2 and RRM3 domains.     

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.     

In vitro splicing of simian virus 40 early pre mRNA.

The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery.     

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.     

In vitro angiogenesis in fibrin matrices containing fibronectin or hyaluronic acid.

Angiogenesis involved numerous interactions between extracellular matrix and endothelial cells which may exhibit changes in actin filament distribution. Using an in vitro model, capillary endothelial cells were grown in fibrin matrix containing fibronectin or hyaluronic acid. Actin filament distribution, nucleus localization and cell morphology were observed. Preliminary study showed the formation of tube-, branche- and capillary-like structures within fibrin. In the presence of both fibrin and fibronectin, cells with actin filament stress fibers were more spreading than those in fibrin. In the presence of hyaluronic acid, tubes were limited in extension into the fibrin. In addition, the study of co-localization of nucleus and actin filaments showed different cell behaviours. Migratory cells seem to arrange in parallel to each other and a capillary-like structure may be formed at the proximal extremity of this cell pattern.     

The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo.

We describe a new approach to elucidate the role of 3'-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 3'-end-processing machinery, is sufficient for the inhibition of not only 3'-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3'-end processing, thereby establishing that 3'-end processing is required for the splicing of a 3' terminal intron in vivo. Because the NS1A protein causes a global suppression of 3'-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 3'-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3'-end formation. In contrast, the 3'-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.     

Alternative splicing of fibronectin: three variants, three functions.

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. The functional complexity is paralleled by structural diversity in that multiple forms of FN are generated by cell type-specific alternative splicing. In the rat, up to 12 different combinations of the three alternatively spliced segments (EIIIA, EIIIB, and the V region) are produced. What effects do these segments have on FN function? Recently, progress has been made in the identification of specific activities for the three Variants of the V region, V120, V95, and V0. FN-mediated cell adhesion, FN synthesis and secretion, and incorporation into blood clots are differentially affected by these isoforms. These results suggest that cellular behavior is modulated by environmental cues provided by different types and proportions of alternatively spliced FN variants.     

Disulfide-bonded dimerization of fibronectin in vitro.

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.     

trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.