Complex splicing control of the human Thrombopoietin gene by intronic G runs

The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1–4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3′ splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3–5 guanosines (namely, G1–G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7–G10 work in a combinatorial way to control the selection of the proper 3′ splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3′ splice site so providing functional evidence that this factor is involved in the selection of the authentic 3′ splice site of THPO IVS2.     

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.     

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5′ proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3′ hepatic control region, derived from a region ∼18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3′ splice acceptor sites causing deletion of cloned 5′ untranslated mRNA sequences and, in some cases, deletion of the 5′ end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3′ splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1–exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.     

At Least One Intron Is Required for the Nonsense-Mediated Decay of Triosephosphate Isomerase mRNA: a Possible Link between Nuclear Splicing and Cytoplasmic Translation

Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3′-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3′-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3′-most intron from pre-mRNA “marks” the 3′-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5′ untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.     

Does distance matter? Variations in alternative 3′ splicing regulation

Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5′ or 3′ splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3′ splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3′ splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3′ splice sites from constitutive 3′ splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites.     

CA- and Purine-Rich Elements Form a Novel Bipartite Exon Enhancer Which Governs Inclusion of the Minute Virus of Mice NS2-Specific Exon in Both Singly and Doubly Spliced mRNAs

The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5′-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5′ and 3′ regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3′ splice site upstream (i.e., the exon 5′ terminus) or a strong 5′ splice site downstream (i.e., the exon 3′ terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3′ splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3′ splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5′ splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.     

The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly with egfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of the egfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.     

Oriented Scanning Is the Leading Mechanism Underlying 5′ Splice Site Selection in Mammals

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection.