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The 70K subunit of U1 snRNP

Transient transfection of the U1 snRNP 70K protein into COS cells induces nuclear reorganization and redistribution of the splicing factor SC-35; hnRNP proteins are not affected. Correspondingly, splicing and nucleocytoplasmic transport of a coexpressed mRNA substrate is reduced by overexpression of U1-70K. The carboxy-terminal portion of U1-70K-encompassing repeats of Arg/Ser, Arg/Glu, and Arg/Asp localizes to the nucleus independently of U1 RNA and is responsible for these inhibitory effects. This region of U1-70K contains amino acid residues similar to those found in splicing factors SC-35, U2AF, su(wa), and in other SR proteins, suggesting that U1-70K protein may serve as a focus of assembly for functional components of the splicing/transport machinery. These findings are compatible with models that propose that direct interaction between U1-70K and SR proteins plays a regulatory role in early events of spliceosome assembly (Romac, 1995).

A monoclonal antibody (mAb) to the U1 snRNP component U1 70K recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K has been previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus the novel mAb U1 70K antigens have been designated as the RD family. Comparison of mAb U1 70K with a recently characterized antibody (mAb 16H3) whose epitope is a repeating R/D or R/E motif, shows that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors (Staknis, 1995).

The S. cerevisiae SNP1 gene encodes the U1 snRNP specific protein U1-70K. The RRM and glycine rich domains in U1-70K proteins are well conserved from yeast to metazoan, with over 80% amino acid similarity. Yeast strains in which the SNP1 gene is disrupted are viable, but exhibit greatly increased doubling rates and severe temperature sensitivities. In addition, snp1-null strains are defective in nuclear, pre-mRNA splicing. Deletion alleles of SNP1 have been tested for their ability to complement these phenotypes. The highly conserved RRM and glycine rich domains of Snp1 are not required for complementation of the snp1-null growth or splicing defects. However, the amino terminal domain of Snp1, which is not highly conserved, is necessary and sufficient for complementation (Hilleren, 1995).

Expression of the recombinant human U1-70K protein in COS cells results in its rapid transport to the nucleus, even when binding to U1 RNA is debilitated. Deletion analysis of the U1-70K protein reveals the existence of two segments of the protein that were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein that is capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif associated NLS demonstrates that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus (Romac, 1994).

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP have been studied. Both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrate that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lack the U1-70K RNP-80 motif and do not bind naked U1 RNA, associate stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain is specific for the U1 snRNP; stable binding is not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins is supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, are detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins (Nelissen, 1994).

Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that trigger apoptotic cell death when stimulated with ligand or cross-linking antibody -- the mechanism involved has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor than can inhibit interleukin-1 beta converting enzyme (ICE) and ICE-like proteases (See Drosophila Caspase-1). Expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways. The 70-kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage is inhibited by expression of CrmA, but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway (Tewari, 1995).

Analysis of protein C, the C component of U1 snRNP

The U1 small nuclear ribonucleoprotein (snRNP) contains three specific proteins denoted 70K, A and C, in addition to the common proteins. Protein C is involved in the binding of U1 snRNP to the 5' splice site of a pre-mRNA. Unlike proteins A and 70K, U1-C lacks an RNA binding domain (RNP-80 motif) and does not appear to bind directly to U1 snRNA. At the amino terminal end, however, protein C contains a zinc finger-like structure of the CC-HH type found in transcription factor TF IIIA. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles: (1) deletion analysis of protein C shows that the N-terminal 45 amino acids are sufficient for binding to U1 snRNPs; (2) there is modification of the cysteine residues in the N-terminal domain with N-ethylmaleimide and (3) single point mutations of the cysteines and histidines contributing to the putative zinc finger abolish the binding of protein C to U1 snRNPs. Interestingly, unlike the proteins U1-A and U1-70K the U1-C protein is unable to bind to naked U1 snRNA. It is shown however, that protein C does not bind to the known protein constituents of the U1 particle without the U1 snRNA being present. These data indicate that the binding of protein C to U1 snRNP is dependent on the presence of both the U1 snRNA and one or more of the U1 snRNP proteins (Nelissen, 1991).

The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif that is required for the binding of the U1C protein to the U1 snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study it is demonstrated that the human U1C protein is able to form homodimers as well. U1C homodimers are found in three cases: when the human U1C protein is expressed in Escherichia coli; when immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when the yeast two hybrid system is used. Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system reveal that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization (Gunnewiek, 1995).

The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. The third U1 snRNP-specific protein, U1C, passively enters the nucleus. In both X. laevis oocytes and cultured HeLa cells, mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP (Klein Gunnewiek, 1997).

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, transcripts encoding a myc-tagged C protein were injected into amphibian oocytes. The distribution of protein translated from the injected RNA is essentially the same in continuous and pulse-label experiments. In both cases the C protein localizes within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that causes truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localizes normally in the germinal vesicle and can be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own, there to associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localizes within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs (Jantsch, 1992).

The U1 small nuclear ribonucleoprotein particle (snRNP) has an important function in the early formation of the spliceosome, the multicomponent complex in which pre-mRNA splicing takes place. The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. The third U1 snRNP-specific protein, U1C, passively enters the nucleus. In both X. laevis oocytes and cultured HeLa cells, mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP (Gunnewiek, 1997).

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals -- this engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. Recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. It is proposed that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway (Du, 2002).

Interaction of u1snRNP with nuclear cap-binding complex

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is as yet only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has been shown to play a role in pre-mRNA splicing. CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. This function of CBC is conserved in humans and yeast (Lewis, 1996).

U snRNP assembly in yeast involves the La protein

In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. The La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP (Xue, 2000).

These results reveal that the role of the yeast La protein is not limited to the biogenesis of RNA polymerase III transcripts. Instead, Lhp1p plays a more general role in small RNA biogenesis. Consistent with the preference of La proteins for RNAs terminating in UUUOH, each of the pre-U RNAs ends in a run of uridylates. While the mechanism by which snRNA 3' ends are generated in S. cerevisiae is not fully understood, strains defective in the enzyme RNase III exhibit decreased levels of similar U1, U4 and U5 RNA precursors and reduced levels of mature U2 and U5L RNAs. Also, similar pre-U1, pre-U4 and pre-U5 RNAs accumulate in cells containing mutations in several 3' exonucleases. Thus, the pre-U RNAs bound by Lhp1p are most likely to be processing intermediates, generated by RNase III cleavage and subsequent exonuclease digestion (Xue, 2000).

These experiments reveal that the binding of Lhp1p to pre-U RNAs has important consequences for snRNP assembly. As only the pre-U4 RNA is an efficient substrate for Smd1p binding in extracts, the major role of Lhp1p in U4/U6 snRNP assembly may be to stabilize this RNA, thus facilitating Sm (the core proteins of snRNPs) protein binding. Since cells that contain wild-type SMD1 do not require Lhp1p, Sm protein binding may normally be sufficiently rapid such that prolonged stabilization of the precursor is unnecessary. Since addition of Lhp1p to lhp1::LEU2 extracts results in a small increase in Smd1p binding, Lhp1p may also directly facilitate assembly of pre-U4 RNAs into snRNPs by assisting RNA folding, stabilizing RNA structure or interacting with snRNP proteins. Moreover, as Lhp1p has a small effect on Smd1p binding in wild-type extracts, other situations that reduce the efficiency of U snRNP assembly could cause cells to require Lhp1p. In any case, the finding that Lhp1p facilitates U4/U6 snRNP biogenesis supports the hypothesis that Lhp1p functions as a molecular chaperone, i.e. a transiently binding protein, not found in the final assembly, that facilitates the correct fate of newly synthesized RNAs in vivo (Xue, 2000).

Does stabilization of pre-U RNAs by the La protein facilitate U snRNP assembly in higher cells? In vertebrates, binding by Sm proteins to pre-U RNAs occurs in the cytoplasm, and several snRNAs undergo 3' end maturation prior to reimport into the nucleus. As the human La protein binds a cytoplasmic population of U1 RNAs that are longer than mature U1 RNA, the vertebrate protein could function in the cytoplasm to facilitate assembly of pre-U1 RNA into snRNPs. However, the mammalian La protein has not been described as binding U2, U4 or U5 RNA precursors, making analogies difficult. Moreover, as a cytoplasmic phase in snRNP assembly has not been demonstrated in S.cerevisiae, U snRNPs could assemble entirely within the nucleus in this yeast. Consistent with nuclear assembly, pre-U4 RNAs (which are confined to the cytoplasm in mammalian cells) assemble into U4/U6·U5 tri-snRNPs in yeast. Interestingly, the SMN protein (the spinal muscular atrophy gene product), which binds Sm core proteins and is required for snRNP assembly in the vertebrate cytoplasm, has not been identified in S. cerevisiae. Thus, binding by Lhp1p to pre-U RNAs in the nucleus of budding yeast may substitute for the cytoplasmic role played by SMN in other organisms (Xue, 2000).

SMN complex and the assembly of the spliceosome

The small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U5 and U4/U6 are major components of the spliceosome. Each snRNP consists of one snRNA (U1, U2, U5 or U4/U6), an Sm protein core and a set of proteins that are specific to individual snRNAs. The Sm proteins B/B', D1, D2, D3, E, F and G are common to all spliceosomal snRNPs and are arranged into a seven-membered ring that assembles for each snRNA on a consensus sequence motif (PuAU4- 6GPu) called the Sm site, where Sm refers to a series of autoantigens defined using autoantibodies. This assembly process takes place in the cytoplasm shortly after the nuclear export of nascent snRNAs. After the formation of the Sm core, the 7-methyl guanosine (m7G) cap of these snRNAs is hypermethylated to become a 2,2,7-trimethyl guanosine (m3G or TMG). Properly assembled Sm core, cap hypermethylation and 3' end processing of the snRNAs are required for the subsequent nuclear import of the snRNPs, where they function in splicing (Yong, 2002 and references therein).

Important and unexpected insights into the process of snRNP assembly came from studies on the function of the survival of motor neurons (SMN) protein. SMN is the protein product of the spinal muscular atrophy (SMA) disease gene. SMA is a severe neuromuscular disease characterized by degeneration of motor neurons in the spinal cord. Over 98% of SMA patients have deletions or mutations of the telomeric copy of the gene (SMN1) and produce markedly reduced levels of the SMN protein. SMN is part of a large multiprotein complex that also contains Gemin2, the DEAD box RNA helicase Gemin3, Gemin4 and several additional as yet uncharacterized proteins. The SMN complex is present in both the nucleus and the cytoplasm of all metazoan cells, suggesting that it may have multiple functions in cells. Most lines of evidence indicate that the SMN complex functions in the assembly and metabolism of various RNPs, including snRNPs, snoRNPs, and the machineries that carry out transcription and pre-mRNA splicing (Yong, 2002 and references therein). The process of snRNP assembly can be most readily studied in Xenopus oocytes, where specific reagents and intermediates can be microinjected into either the nucleus or the cytoplasm and where dissection of nuclear and cytoplasmic fractions can be readily performed. Such experiments have revealed that the SMN complex associates with spliceosomal U1, U4 and U5 snRNAs in the cytoplasm. Antibodies against components of the SMN complex microinjected into Xenopus oocytes also inhibit the assembly of snRNPs, indicating that the SMN complex plays a crucial role in the biogenesis of snRNPs. In addition, overexpression of a dominant-negative SMN mutant blocks snRNP assembly in the cytoplasm of somatic cells, suggesting a general function for the SMN complex in the cytoplasmic phase of U snRNAs biogenesis. Recent studies have further demonstrated that the SMN complex is necessary for assembly of U1 snRNP in Xenopus egg extracts (Yong, 2002 and references therein).

The capacity of the SMN complex to associate with and mediate the assembly of snRNPs is probably due, at least in part, to interactions between the SMN complex and snRNP proteins. Several of the components of the SMN complex interact directly with Sm proteins. In particular, SMN binds avidly to RG-rich C-terminal domains that are found in the Sm proteins B, D1 and D3, whereas several SMN mutants found in SMA patients are defective in Sm protein binding. Importantly, SMN binds preferentially to the RG domains of D1 and D3 after arginines in specific positions are converted to symmetric dimethylarginines (sDMAs). Thus, arginine dimethylation has a key role in the protein substrate recognition by the SMN complex, and RNP assembly is likely to be regulated by arginine methylation (Yong, 2002 and references therein).

To serve in snRNP assembly, the SMN-Sm protein complex must also recruit the snRNAs. The binding of U1 snRNA, an abundant and high-avidity substrate, to the SMN complex has been investigated. The binding is sequence-specific and is mediated by the loop of stem- loop 1 domain (SL1) of U1 snRNA. SL1 is both necessary and sufficient for the interaction with the SMN complex in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. The interaction between the SMN complex and U1 snRNA is required for U1 snRNP assembly and thus mediates the function of the SMN complex in U1 snRNP biogenesis (Yong, 2002).

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