Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K

We have investigated the structure of the small nuclear RNP (snRNP) U1 by combining EM of complete and partially protein-deficient particles with immunoelectron microscopy employing mAbs against known components of the U1 snRNP. It was found that the two main protuberances of this particle can be identified with the U1-specific proteins A and 70K. The 70K protuberance is the one lying closer to the 5' terminus of the snRNA, as identified by its 5'-terminal m3G cap. The round-shaped main body of U1 snRNP represents its core RNP domain containing the common snRNP proteins. Functional implications of these results are discussed. Our results may also point to the physical basis for the production of autoantibodies directed against specific groups of snRNP proteins. The physical grouping of the common proteins (Sm epitopes) and the specific proteins (RNP epitopes) could result in one or the other being presented to the immune system as is the case in patients suffering from SLE or MCTD, respectively.     

G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site

Highly conserved G runs, G1M2 and ISE, regulate the proteolipid protein (PLP)/DM20 ratio. We have investigated recruitment of U1 small nuclear ribonuclear protein (snRNP) by G1M2 and ISE and examined the effect of splice site strength, distance, and context on G run function. G1M2 is necessary for initial recruitment of U1snRNP to the DM20 5' splice site independent of the strength of the splice site. G1M2 regulates E complex formation and supports DM20 splicing when functional U1snRNP is reduced. By contrast, the ISE is not required for the initial recruitment of U1snRNP to the PLP 5' splice site. However, in close proximity to either the DM20 or the PLP 5' splice site, the ISE recruits U1snRNP to both splice sites. The ISE enhances DM20 splicing, whereas close to the PLP 5' splice site, it inhibits PLP splicing. Splicing enhancement and inhibition are mediated by heterogeneous nuclear ribonuclear protein (hnRNP)H/F. The data show that recognition of the DM20 5' splice site depends on G run-mediated recruitment of U1snRNA, whereas a complex interaction between the ISE G runs, context and position determines the functional outcome on splicing. The data suggest that different mechanisms underlie G run-mediated recognition of 5' splice sites and that context and position play a critical role.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

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.     

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.     

Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins

The 5' ends of U1, U2, U3, U4, and U5 small nuclear RNAs (snRNA) are capped by a structure which contains N2,N2-7-trimethylguanosine (m2,2,7 G). m2,2,7 G was used as hapten to raise antibodies in rabbits, and these antibodies were linked to Sepharose. When deproteinized RNA was passed through this antibody column, these snRNA species were retained by the column. Conversely, 4 S, 5 S, 5.8 S, U6, and 7 S RNA, whose 5' termini do not contain m2,2,7 G, were not recognized. After a nuclear extract was loaded on the column, U1 RNA and some U2 RNA were retained. Therefore, the 5' ends of at least U1 RNA are accessible when this RNA species is in small nuclear ribonucleoprotein particle (snRNP) form. This is of interest, since it has been proposed that the 5' terminus sequence of U1 RNA may hybridize with splice junctions in heterogeneous nuclear ribonucleoprotein particles (hnRNP) during mRNA splicing. The retention of m2,2,7 G-containing RNA species by these antibodies is not due to association of snRNAs or snRNPs with heterogeneous nuclear RNA (hnRNA) or hnRNP (and antibody recognition of 7-monomethylguanosine residues in hnRNA), since the reaction still occurs after removal of hnRNA or hnRNP by sucrose gradient centrifugation.     

Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP

We describe the electron microscopic investigation of purified U4/U6 snRNPs from human and murine cells. The U4/U6 snRNP exhibits two morphological features, a main body approximately 8 nm in diameter and a peripheral filamentous domain, 7-10 nm long. Two lines of evidence suggest that the peripheral domain may consist of RNA and to contain U6 RNA as well as the 5' portion of U4 RNA. (a) Separation of the U4/U6 snRNA interaction regions from the core domains by site-directed cleavage of the U4 snRNA with RNase H gave filament-free, globular core snRNP structures. (b) By immuno and DNA-hybridization EM, both the 5' end of U4 and the 3' end of U6 snRNA were located at the distal region of the filamentous domain, furthest from the core. These results, together with our observation that the filamentous U4/U6 domain is often Y shaped, correlate strikingly with the consensus secondary structure proposed by Brow and Guthrie (1988. Nature (Lond.), 334:213-218), where U4 and U6 snRNA are base paired in such a way that two U4/U6 helices together with a stem/loop of U4 snRNA make up a Y-shaped U4/U6 interaction domain.     

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in 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.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

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.     

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes

Electrophoretic separation of ribonucleoprotein particles in a nondenaturing gel was used to analyze the splicing of mRNA precursors. Early in the reaction, a complex formed consisting of the U2 small nuclear ribonucleoprotein particle (snRNP) bound to sequences upstream of the 3' splice site. This complex is modeled as a precursor of a larger complex, the spliceosome, which contains U2, U4/6, and U5 snRNPs. Conversion of the U2 snRNP-precursor RNA complex to the spliceosome probably involves binding of a single multi-snRNP particle containing U4/6 and U5 snRNPs. The excised intron was released in a complex containing U5, U6, and probably U2 snRNPs. Surprisingly, U4 snRNP was not part of the intron-containing complex, suggesting that U4/6 snRNP disassembles and assembles during splicing. Subsequently, the reassembled U4/6 snRNP would associate with U5 snRNP and participate in de novo spliceosome formation. U1 snRNP was not detected in any of the splicing complexes.     

Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.     

Plant small nuclear RNAs. V. U4 RNA is present in broad bean plants in the form of sequence variants and is base-paired with U6 RNA.

U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles. It occurs in broad bean in the form of a number of sequence variants. Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long. Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae. Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half. The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far. Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules. A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA. This is the first report on the structure of a plant U4 RNA.