Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types. To study CT specific processing in Hela cell nuclear extracts we have used precursor RNAs corresponding to the exon 3 to exon 4 region containing only CT specific processing signals. The results revealed the usage of a uridine residue 23 nucleotides upstream of the 3' splice site as the major site of lariat formation in CT specific splicing. The implications of this finding for the alternative, tissue specific processing of the CALC-I pre-mRNA and for branch point selection in general are discussed.     

In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract

An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.     

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.     

Bridging sulfur substitutions in the analysis of pre-mRNA splicing

Accurate excision of intervening sequences (introns) from messenger RNA precursors is accomplished by a very large and complicated ribonucleoprotein complex called the spliceosome. Elucidating the mechanisms of the two phosphotransesterification reactions that result in intron removal is important for our understanding of the molecular evolution of early genetic systems, as well as our knowledge of contemporary eukaryotic gene expression. The functional consequences of systematic alterations in the reactive groups can be invaluable for understanding catalytic mechanisms, especially for enzymes, such as the spliceosome, whose size and complexity place them beyond the reach of crystallographic and spectroscopic analysis. One type of modification that can be incorporated into a scissile phosphate linkage is the phosphorothiolate, in which a bridging phosphate oxygen is substituted with sulfur. Phosphorothiolate substitutions can be used to detect metal ion-ligand interactions by a "metal specificity switch" strategy. I review recent advances in the synthesis, incorporation, and manipulation of nucleoside phosphorothiolates (with an emphasis on 3'-S-phosphorothiolates), and describe their utility in the study of pre-mRNA splicing.     

Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.     

Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA.

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.     

Statistical analysis of mammalian pre-mRNA splicing sites.

222 donor and 222 acceptor (including 206 pairs) non-homologous splicing sites were studied. Well known features of these were confirmed and some novel observations were made. It is (1) cCAGGGag signal in (-60)-(-58) region of acceptor sites; (2) strong complementarity between regions (-69)-(-55) and (-36)-(-22) of some of the acceptor sites, and (3) small but statistically significant correlation between discrimination energies of corresponding donor and acceptor sites.     

Inhibition of nuclear pre-mRNA splicing by antibiotics in vitro.

A number of antibiotics have been reported to disturb the decoding process in prokaryotic translation and to inhibit the function of various natural ribozymes. We investigated the effect of several antibiotics on in vitro splicing of a eukaryotic nuclear pre-mRNA (beta-globin). Of the eight antibiotics studied, erythromycin, Cl-tetracycline and streptomycin were identified as splicing inhibitors in nuclear HeLa cell extract. The K(i) values were 160, 180 and 230 microm, respectively. Cl-tetracycline-mediated and streptomycin-mediated splicing inhibition were in the molar inhibition range for hammerhead and human hepatitis delta virus ribozyme self-cleavage (tetracycline), of group-I intron self-splicing (streptomycin) and inhibition of RNase P cleavage by some aminoglycosides. Cl-tetracycline and the aminocyclitol glycoside streptomycin were found to have an indirect effect on splicing by unspecific binding to the pre-mRNA, suggesting that the inhibition is the result of disturbance of the correct folding of the pre-mRNA into the splicing-compatible tertiary structure by the charged groups of these antibiotics. The macrolide, erythromycin, the strongest inhibitor, had only a slight effect on formation of the presplicing complexes A and B, but almost completely inhibited formation of the splicing-active C complex by binding to nuclear extract component(s). This results in direct inhibition of the second step of pre-mRNA splicing. To our knowledge, this is the first report on specific inhibition of nuclear splicing by an antibiotic. The functional groups involved in the interaction of erythromycin with snRNAs and/or splicing factors require further investigation.     

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.