An additional long-range interaction in human U1 snRNA.

We present evidence for the existence of an additional long-range interaction in vertebrate U1 snRNAs. By submitting human U1 snRNP, HeLa nuclear extracts, authentic human or X. laevis in vitro transcribed U1 snRNAs to RNase V1, a nuclease specific for double-stranded regions, cleavages occurred in the sequence psi psi ACC (positions 5-9) residing in the 5' terminal region of the RNA. The RNase V1 sensitive region is insensitive to single-stranded probes, something unexpected knowing that it was considered single-stranded in order to base-pair to pre-mRNA 5' splice site. We have identified the sequence GGUAG (positions 132-136) as the only possible 3' partner. Mutants, either abolishing or restoring the interaction between the partners, coupled to an RNase V1 assay, served to substantiate this base-pairing model. The presence of this additional helix, even detected in nuclear extracts under in vitro splicing conditions, implies that a conformational change must occur to release a free U1 snRNA 5' end.     

Who's on first? The U1 snRNP-5' splice site interaction and splicing

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.     

A short 5' splice site RNA oligo can participate in both steps of splicing in mammalian extracts.

A short 5' splice site RNA oligonucleotide (5'SS RNA oligo) undergoes both steps of splicing when a second RNA containing the 3' splice site region (3'SS RNA) is added in trans. This trans-splicing reaction displays the same 5' and 3' splice site sequence requirements as cis-splicing of full-length pre-mRNA. The analysis of RNA-snRNP complexes formed on each of the two splice site RNAs is consistent with the formation of partial complexes, which then associate to form the complete spliceosome. Specifically, U2 snRNP bound to the 3'SS RNA associates with U4/U5/U6 snRNP bound to the 5'SS RNA oligo. Thus, as expected, trans-splicing depends on the integrity of U2, U4, and U6 snRNAs. However, unlike cis-splicing, trans-splicing is enhanced when the 5' end of U1 snRNA is blocked or removed or when the U1 snRNP is depleted. Thus, the early regulatory requirement for U1 snRNP, which is essential in cis-splicing, is bypassed in this trans-splicing system. This simplified trans-splicing reaction offers a unique model system in which to study the mechanistic details of pre-mRNA splicing.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts. From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.     

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end

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.     

Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing

Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation.     

A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates human immunodeficiency virus type 1 rev, env, vpu, and nef gene expression

The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.     

Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.     

Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing.

Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop Ila region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.     

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.     

A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5'' cleavage site.

We analyzed the effects of suppressor mutations in the U1 snRNA (SNR19) gene from Saccharomyces cerevisiae on the splicing of mutant pre-mRNA substrates. The results indicate that pairing between U1 snRNA and the highly conserved position 5 (GTATGT) of the intron occurs early in spliceosome assembly in vitro. This pairing is important for efficient splicing both in vitro and in vivo. However, pairing at position 5 does not appear to influence 5' splice site selection in vivo, indicating that the previously described U1 snRNA:5' splice junction base pairing interaction is not sufficient to define the 5' cleavage site.     

In vitro splicing of mRNA precursors: 5'' cleavage site can be predicted from the interaction between the 5'' splice region and the 5'' terminus of U1 snRNA.

Combinations of different mutations within the 5' splice region of the rabbit beta-globin large intron were analyzed for their effect on in vitro splicing. Based upon the complementarity of the 5' splice region to the 5' terminal region of the U1 snRNA, the exact location of the 5' cleavage site of different mutants could be predicted and was experimentally confirmed. These findings add further strong support to the hypothesis (1) that the exact location of the 5' cleavage site in pre-mRNA splicing of higher eukaryotes is determined by the overall 5' splice region via the complementarity to the 5' end of the U1 snRNA, and not by the strongly conserved GU dinucleotide.     

Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro

We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract. SF3 is a factor that can be preferentially inactivated by moderate heat treatment. Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography. SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation.     

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.     

Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly

HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3' splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable preslicing complex formation is independent of the U1 snRNA-5' splice site interaction.     

The 5''-terminal sequence of U1 RNA complementary to the consensus 5'' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.     

Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.