U1 snRNPs Research Articles

Recognition of the 5' splice site by the spliceosome.

The splicing of nuclear pre-mRNAs is catalyzed by a large, multicomponent ribonucleoprotein complex termed the spliceosome. Elucidation of the molecular mechanism of splicing identified small nuclear RNAs (snRNAs) as important components of the spliceosome, which, by analogy to the self-splicing group II introns, are implicated in formation of the catalytic center. In particular, the 5' splice site (5'SS) and the branch site, which represent the two substrates for the first step of splicing, are first recognized by U1 and U2 snRNPs, respectively. This initial recognition of splice sites is responsible for the global definition of exons and introns, and represents the primary target for regulation of splicing. Subsequently, pairing interaction between the 5'SS and U1 snRNA is disrupted and replaced by a new interaction of the 5'SS with U6 snRNA. The 5'SS signal contains an invariant GU dinucleotide present at the 5' end of nearly all known introns, however, the mechanism by which the spliceosome recognizes this element is not known. We have identified and characterized a specific UV light-induced crosslink formed between the 5'SS RNA and hPrp8, a protein component of U5 snRNP in the spliceosome that is likely to reflect a specific recognition of the GU dinucleotide for splicing. Because recognition of the 5'SS must be linked to formation of the catalytic site, the identification of a specific and direct interaction between the 5'SS and Prp8 has significant implications for the role of this protein in the mechanism of mRNA splicing.

Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing.

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.

Coilin can form a complex with the U7 small nuclear ribonucleoprotein.

Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 microm or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 microm. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50-100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription.

WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: the proline glycine and methionine-rich motif.

Pre-mRNA splicing requires the bridging of the 5' and 3' ends of the intron. In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP. Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP's), each of which contains one or more of a special class of tyrosine-rich WW domains. Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40. We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB', and the branchpoint binding protein SF1/mBBP. Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex.

Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA.

This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.

Regulation of alternative polyadenylation by U1 snRNPs and SRp20.

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3'-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3'-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3'-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3' and 5' splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3'-terminal exons and perhaps in the communication between polyadenylation and splicing.

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point.

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2

Background: The dynamic rearrangements of RNA structure which occur during pre-mRNA splicing are thought to be mediated by members of the DExD/H-box family of RNA-dependent ATPases. Although three DExD/H-box splicing factors have recently been shown to unwind synthetic RNA duplexes in purified systems, in no case has the natural biological substrate been identified. A duplex RNA target of particular interest is the extensive base-pairing interaction between U4 and U6 small nuclear RNAs. Because these helices must be disrupted to activate the spliceosome for catalysis, this rearrangement is believed to be tightly regulated in vivo.Results: We have immunopurified Brr2, a DEIH-box ATPase, in a native complex containing U1, U2, U5 and duplex U4/U6 small nuclear ribonucleoprotein particles (snRNPs). Addition of hydrolyzable ATP to this complex results in the disruption of U4/U6 base-pairing, and the release of free U4 and U6 snRNPs. A mutation in the helicase-like domain of Brr2 (brr2-1) prevents these RNA rearrangements. Notably, U4/U6 dissociation and release occur in the absence of exogenously added pre-mRNA.Conclusions: Disruption of U4/U6 base-pairing in native snRNPs requires ATP hydrolysis and Brr2. This is the first assignment of a DExD/H-box splicing factor to a specific biological unwinding event. The unwinding function of Brr2 can be antagonized by the annealing activity of Prp24. We propose the existence of a dynamic cycle, uncoupled from splicing, that interconverts free and base-paired U4/U6 snRNPs.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing.

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.

Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing.

The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes. We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking. Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing. Larger insertions or deletions allowed the first step but progressively inhibited the second step. Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon.

Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins.

The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5' splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination.

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL is maintained by a positive autoregulatory feedback loop in which Sxl protein insures its continued synthesis by binding to Sxl pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. We also show that female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu which control female-specific processes in the ovary.

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.

Dynamics of the U1 small nuclear ribonucleoprotein during yeast spliceosome assembly.

U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly. Most spliceosome assembly assays, however, fail to detect the U1 snRNP. Here, I used a new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (delta, beta1, alpha2) formed in vitro. The order of complex formation is deduced to be delta --> beta1 --> alpha2 --> alpha1 --> beta2, the active spliceosome. The delta complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP. There are two forms of delta: a major one, deltaun, unstable to competitor RNA; and a minor one, deltacommit, committed to the splicing pathway. The other complexes are formed in the presence of ATP and contain the following snRNPs: beta1, the pre-spliceosome, has both U1 and U2; alpha2 has all five, however, U1 is reduced compared with the others; and alpha1 and beta2 have U2, U5, and U6. Prior work by others suggests that U1 is "handing off" the 5' splice site region to the U5 and U6 snRNPs before splicing begins. The reduced levels of U1 snRNP in the alpha2 complex suggests that the handoff occurs during formation of this complex.

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.

Human anti-nuclear ribonucleoprotein antigen autoimmune sera contain a novel subset of autoantibodies that stabilizes the molecular interaction of U1RNP-C protein with the Sm core proteins.

Anti-Sm Abs recognize Sm core proteins B'/B, D, E, F, and G, shared by U1, U2, U4-6, and U5 small nuclear ribonucleoproteins (snRNPs), while anti-nuclear ribonucleoprotein Ag (nRNP) Abs recognize the U1 RNP-specific 70K, A, and C proteins. However, although the autoimmune response to U1 snRNPs involves all components of the particle, not all are recognized equally. For example, all human anti-nRNP sera contain Abs against native U1-C, in contrast to their absence in MRL/lpr mice. In this study, autoantibody recognition of native U1 snRNPs was investigated by dissociating the particle into four components (U1-70K, U1-A, U1-C, and the Sm core particle) using 1 M MgCl2 or ribonuclease treatment. As expected, human anti-Sm and MRL/lpr sera immunoprecipitated only the Sm core proteins, and human anti-nRNP/Sm sera immunoprecipitated the Sm core proteins plus U1-C under both conditions. However, although human anti-nRNP sera immunoprecipitated U1-C when U1 snRNPs were dissociated before Ab binding, they unexpectedly immunoprecipitated the Sm core proteins when Abs were bound before dissociation. This apparent paradox was explained by the stabilizing effects of anti-nRNP sera on interactions of U1-C with the Sm core particle. All human anti-nRNP sera contained high levels of autoantibodies that prevent dissociation of U1-C from the U1 snRNP. These Abs were absent in MRL/lpr mice. Human autoimmune sera may prevent dissociation by recognizing the quaternary structure of the U1-C-Sm core protein complex or by altering its conformation. Stabilization of U1 snRNPs by autoantibodies could influence Ag processing and presentation, possibly with important effects on the development of autoimmunity to U1 snRNPs.

Analysis of genes for human snRNP Sm-D1 protein and identification of the promoter sequence which shows segmental homology to the promoters of Sm-E and U1 snRNA genes

The Sm core proteins of U1, U2, U4/U6 and U5 snRNPs include B(B1), B′(B2), N(B3), D1, D2, D3, E, F and G polypeptides. We have isolated genomic clones encoding the Sm-D1 protein using the Sm-D1 cDNA as probe. Southern blotting and DNA sequencing analysis of these clones revealed the presence of an Sm-D1 multigene family in the human genome. Three gene members have been identified. Two of the genes are without introns and contain mutations compared to the cDNA sequence. They appear to be processed pseudogenes. The third gene, termed SNRPD1, shares 100% identity to the cDNA sequence including both 5′- and 3′-untranslated regions ( UTR); it contains three introns. Analysis of the 5′-flanking region of the SNRPD1 gene revealed promoter activity, suggesting this is the functional gene that encodes the Sm-D1 protein. The promoter activity was localized in a 0.38 kb PstI fragment using CAT reporter gene fusion assays. Addition of an SV40 enhancer element did not enhance the transcription directed by that fragment. Sequence comparison of the 0.38 kb promoter sequence with the promoters of the Sm-E gene and U1 snRNA genes revealed several homologous motifs, suggesting that genes encoding the snRNP components may be coordinately regulated.

In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes.

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.

The C-group Heterogeneous Nuclear Ribonucleoprotein Proteins Bind to the 5′ Stem-Loop of the U2 Small Nuclear Ribonucleoprotein Particle

The C-group heterogeneous nuclear ribonucleoprotein (hnRNP) proteins bind to nascent pre-messenger RNA. In vitro studies have indicated that the C hnRNP proteins bind particularly strongly to the intron polypyrimidine tract of pre-mRNA and may be important for pre-mRNA splicing. In addition, there is evidence that the interaction of the C hnRNP proteins with pre-mRNA is facilitated by the U1 and U2 small nuclear RNPs (snRNPs). In the present study, we have uncovered another feature of the C hnRNP proteins that may provide a unifying framework for these previous observations; the C hnRNP proteins bind to the 5' stem-loop of the U2 snRNP. This was detected by incubating human 32P-labeled U2 snRNP in micrococcal nuclease-treated HeLa nuclear extracts, followed by UV-mediated protein-RNA cross-linking, which revealed that C hnRNP proteins were cross-linked to 32P-nucleotides in the U2 snRNP. In similar experiments, no cross-linking of C hnRNP proteins to 32P-labeled U1 or U4 snRNPs was observed. The observed cross-linking of C hnRNP proteins to U2 snRNP was efficiently competed by excess U2 RNA and by poly(U) but not by poly(A). No competition was observed with an RNA molecule comprising U2 nucleotides 105-189, indicating that the C hnRNP protein interactive regions of U2 RNA reside solely in the 5' half of the molecule. Oligodeoxynucleotide-mediated RNase H cleavage experiments revealed that a 5' region of U2 RNA including nucleotides 15-28 is essential for the observed C hnRNP protein cross-linking. C hnRNP protein cross-linking to U2 snRNP was efficiently competed by a mini-RNA corresponding to the first 29 nucleotides of U2 RNA, whereas no competition was observed with a variant of this mini-RNA in which the UUUU loop of stem-loop I was mutationally configured into a single-stranded RNA by replacing the stem with non-pairing nucleotides. Competition experiments with another mutant mini-U2 RNA in which the UUUU loop was replaced by AAAA indicated that both the UUUU loop and the stem are important for C hnRNP protein cross-linking, a finding consistent with other recent data on the RNA sequence specificity of C hnRNP protein binding.