Fetal liver tissue collected from a nonhuman primate (NHP) baboon model of maternal nutrient reduction (MNR) at four gestational time points (90, 120, 140, and 165 days gestation [dG], term in the baboon is ∼185 dG) was used to quantify MNR effects on the fetal liver transcriptome. 28 transcripts demonstrated different expression patterns between MNR and control livers during the second half of gestation, a developmental period when the fetus undergoes rapid weight gain and fat accumulation. Differentially expressed transcripts were enriched for fatty acid oxidation and RNA splicing-related pathways. Increased RNA splicing activity in MNR was reflected in greater abundances of transcript splice variant isoforms in the MNR group. It can be hypothesized that the increase in splice variants is deployed in an effort to adapt to the poor in utero environment and ensure near-normal development and energy metabolism. This study is the first to study developmental programming across four critical gestational stages during primate fetal liver development and reveals a potentially novel cellular response mechanism mediating fetal programming in response to MNR.

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3′ splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a “labile” interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then “locked in” by specific binding of I-AniI to its intron interaction site.

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5′ splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5′ splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5′ end, suggesting that other factors than the U1 snRNA are involved in 5′ splice site recognition. The importance of a consecutive complementarity between the 5′ splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5′ splice site upstream of the env open reading frame. To determine the role of this splice site in the 5′-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5′ splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5′ splice site, SD4, and the free 5′ end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5′ splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions −3 to +8 of the 5′ splice site and all 11 nt constituting the single-stranded 5′ end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3′ splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3′ splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3′ terminal intron residue. In this context, mutation of the conserved 3′ intron terminal G to a C also results in the partial activation of a nearby cryptic 3′ splice site with U as the 3′ terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3′ splice site over the normal 3′ splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6–U57 mutants to influence the fidelity of both branch site and 3′ splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2′,3′ cyclic phosphate and 5′-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNAMet has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2′,3′ cyclic phosphate at the 3′ end of the 5′ exon is converted into the splice junction phosphate forming a 3′,5′-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.

An iterative in vitro splicing strategy was employed to select for optimal 3′ splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3′ end of the randomized insert. Mutating the 3′ terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.

Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3′ splice site AG is supplied by a different RNA molecule than that containing the 5′ splice and branch sites. By varying the time during splicing reactions when the 3′ splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3′ splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.

In most species the 3′ splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3′ splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3′ splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.

The spliceosomal proteins U1A and U2B′′ each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpII and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B′′ to U2hpIV is facilitated by the ancillary protein U2A′, whereas specific binding of U1A to U1hpII requires no cofactor. Here we report that U2A′ enables U2B′′ to distinguish the loop sequence of U2hpIV from that of U1hpII but plays no role in stem sequence discrimination. Although U2A′ can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the ∼500-fold greater affinity of U2A′ for U2B′′. Additional experiments have identified a single leucine residue in U1A (Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1hpII in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B′′ can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A′ to the specificity of U2B′′.