The role of branchpoint-3' splice site spacing and interaction between intron terminal nucleotides in 3' splice site selection in Saccharomyces cerevisiae

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.     

A role for U2/U6 helix Ib in 5' splice site selection

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.     

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.     

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.     

Identification of exon sequences involved in splice site selection

The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons. When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct. Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals. A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold. Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold. Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection.     

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.     

Interaction between the negative regulator of splicing element and a 3' splice site: requirement for U1 small nuclear ribonucleoprotein and the 3' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.     

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.     

Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.     

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

Anatomy of lipase binding sites: the scissile fatty acid binding site

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.     

Compound heterozygosity for an out-of-frame deletion and a splice site mutation in the LAMB3 gene causes nonlethal junctional epidermolysis bullosa

OBJECTIVE: To explore the temporal relation of demyelination and blood-brain barrier breakdown during new lesion formation. BACKGROUND: Conventional MRI appears sensitive for detecting changes due to MS, but may be limited by poor pathologic specificity. By indirectly assessing protons bound to rigid macromolecules, magnetization transfer (MT) imaging may provide information relating to tissue structure and, by inference, myelin integrity. METHODS: Gadolinium contrast-enhanced MRI and MT imaging were performed at weekly intervals for 3 months in three patients with MS. For each enhancing lesion, the largest corresponding area of proton density hyperintensity seen during the study was outlined and magnetization transfer ratio (MTR) calculated at each time point from coregistered calculated MTR images. Lesions greater than 20 mm2, not affected by partial volume effects, and first enhancing after the baseline study were analyzed. Two-dimensional registration software allowed accurate evaluation of MTR in regions both before and after the initial appearance of MS lesions. RESULTS: Mean lesion MTR decreased significantly during the first week of enhancement (29.6 percent units [pu] immediately pre-enhancement versus 28.2 pu at first documented stage of enhancement). No significant MTR reduction was noted before this. CONCLUSION: The lack of observable change in MTR before the first detectable gadolinium enhancement within MS lesions suggests that blood-brain barrier disruption is closely related to, but not preceded by, demyelination.