Essential role for the SMN complex in the specificity of snRNP assembly

The Survival of Motor Neurons (SMN) protein, the product of the spinal muscular atrophy-determining gene, is part of a large macromolecular complex (SMN complex) that functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Using cell extracts and purified components, we demonstrated that the SMN complex is necessary and sufficient to mediate the ATP-dependent assembly of the core of seven Sm proteins on uridine-rich, small nuclear ribonucleic acids (U snRNAs). In vitro experiments revealed strict requirements for ordered binding of the Sm proteins and the U snRNAs to the SMN complex. Importantly, the SMN complex is necessary to ensure that Sm cores assemble only on correct RNA targets and prevent their otherwise promiscuous association with other RNAs. Thus, the SMN complex functions as a specificity factor essential for the efficient assembly of Sm proteins on U snRNAs and likely protects cells from illicit, and potentially deleterious, nonspecific binding of Sm proteins to RNAs.     

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.     

The Prp19p-associated complex in spliceosome activation

During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.     

An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.     

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site

One of the functions of U1 small nuclear ribonucleoprotein (snRNP) in the splicing reaction of pre-mRNA molecules is the recognition of the 5' splice site. U1 snRNP proteins as well as base-pair interactions between U1 snRNA and the 5' splice site are important for the formation of the snRNP-pre-mRNA complex. To determine which proteins are needed for complex formation, the ability of U1 snRNPs gradually depleted of the U1-specific proteins C, A, and 70k to bind to an RNA molecule containing a 5' splice site sequence was studied in a nitrocellulose filter binding assay. The most significant effect was always observed when protein C was removed, either alone or together with other U1-specific proteins; the binding was reduced by 50 to 60%. Complementation of protein C-deficient U1 snRNPs with purified C protein restored their 5' splice site binding activity. These data suggest that protein C may potentiate the base-pair interaction between U1 RNA and the 5' splice site.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.     

Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.     

Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction

The reaction mechanism for self-splicing introns requires the existence of a 5' exon binding site on the intron. Experimental evidence is now presented consistent with the existence of such a binding site by demonstrating efficient and accurate trans-self-splicing of a yeast mitochondrial group II intron. Partial and complete trans-splicing reactions take place in the absence of branch formation, part of the usual pathway of nuclear splicing and group II self-splicing. In addition to indicating the existence of a 5' exon binding site on the intron, the results have mechanistic implications for group II self-splicing and perhaps for nuclear splicing as well.     

Association of TALS developmental disorder with defect in minor splicing component U4atac snRNA

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.     

A computational screen for methylation guide snoRNAs in yeast

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Many of the genes for this snoRNA family have remained unidentified in Saccharomyces cerevisiae, despite the availability of a complete genome sequence. Probabilistic modeling methods akin to those used in speech recognition and computational linguistics were used to computationally screen the yeast genome and identify 22 methylation guide snoRNAs, snR50 to snR71. Gene disruptions and other experimental characterization confirmed their methylation guide function. In total, 51 of the 55 ribose methylated sites in yeast ribosomal RNA were assigned to 41 different guide snoRNAs.     

Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.     

Regulatory role for GTP-binding proteins in endocytosis

Guanosine 5'-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells. Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride. The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors. Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway.     

Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.