Dyskerin, tRNA genes, and condensin tether pericentric chromatin to the spindle axis in mitosis.

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

Role for LSM genes in the regulation of circadian rhythms.

Growing evidence suggests that core spliceosomal components differentially affect RNA processing of specific genes; however, whether changes in the levels or activities of these factors control specific signaling pathways is largely unknown. Here we show that some SM-like (LSM) genes, which encode core components of the spliceosomal U6 small nuclear ribonucleoprotein complex, regulate circadian rhythms in plants and mammals. We found that the circadian clock regulates the expression of LSM5 in Arabidopsis plants and several LSM genes in mouse suprachiasmatic nucleus. Further, mutations in LSM5 or LSM4 in Arabidopsis, or down-regulation of LSM3, LSM5, or LSM7 expression in human cells, lengthens the circadian period. Although we identified changes in the expression and alternative splicing of some core clock genes in Arabidopsis lsm5 mutants, the precise molecular mechanism causing period lengthening remains to be identified. Genome-wide expression analysis of either a weak lsm5 or a strong lsm4 mutant allele in Arabidopsis revealed larger effects on alternative splicing than on constitutive splicing. Remarkably, large splicing defects were not observed in most of the introns evaluated using RNA-seq in the strong lsm4 mutant allele used in this study. These findings support the idea that some LSM genes play both regulatory and constitutive roles in RNA processing, contributing to the fine-tuning of specific signaling pathways.

Regulation of alternative splicing by the core spliceosomal machinery.

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B' self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B' in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.

SMN-independent subunits of the SMN complex. Identification of a small nuclear ribonucleoprotein assembly intermediate.

The survival of motor neurons (SMN) complex is essential for the biogenesis of small nuclear ribonucleoprotein (snRNP) complexes in eukaryotic cells. Reduced levels of SMN cause the motor neuron degenerative disease, spinal muscular atrophy. We identify here stable subunits of the SMN complex that do not contain SMN. Sedimentation and immunoprecipitation experiments using cell extracts reveal at least three complexes composed of Gemin3, -4, and -5; Gemin6, -7, and unrip; and SMN with Gemin2, as well as free Gemin5. Complexes containing Gemin3-Gemin4-Gemin5 and Gemin6-Gemin7-unrip persist at similar levels when SMN is reduced. In cells, immunofluorescence microscopy shows differential localization of Gemin5 after cell stress. We further show that the Gemin5-containing subunits bind small nuclear RNA independently of the SMN complex and without a requirement for exogenous ATP. ATP hydrolysis is, however, required for displacement of small nuclear RNAs from the Gemin5-containing subunits and their assembly into snRNPs. These findings demonstrate a modular nature of the SMN complex and identify a new intermediate in the snRNP assembly process.

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice.

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.

RNAi interference of XPO1 and Sm genes and their effect on the spliced leader RNA in Trypanosoma brucei.

In trypanosomes, trans-splicing is a major essential RNA-processing mechanism that involves the addition of a spliced leader sequence to all mRNAs from a small RNA species, known as the spliced leader RNA (SL RNA). SL RNA maturation is poorly understood and it is not clear where assembly with Sm proteins takes place. In this study, we followed the localization of the SL RNA during knockdown of Sm proteins and XPO1, which in metazoa functions in transport of mRNA and U snRNAs from the nucleus to the cytoplasm. We found that XPO1 has no role in SL RNA biogenesis in wild-type cells, or when the cells are depleted of Sm proteins. During Sm depletion, 'defective' SL RNA lacking cap modification at position +4 first accumulates in the nucleus, suggesting that Sm assembly on SL RNA most probably takes place in this compartment. Only after massive nuclear accumulation is the 'defective' SL RNA exported to the cytoplasm to form SL RNP-C, which may be a route to dispose of SL RNA when its normal biogenesis is blocked.

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E.

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.

Membrane estrogen receptors: genomic actions and post transcriptional regulation.

The primary cellular location of the nuclear estrogen receptor II (nER II) is the plasma membrane. A number of reports that have appeared in the recent past indicate that plasma membrane localized estrogen receptor alpha (ERalpha) also exists. Whether the membrane localized ERalpha represents the receptor that binds to the estrogen responsive element (ERE) remains to be known. The mechanisms that underlie the internalization of nER II (non-activated estrogen receptor, deglycosylated) have been identified to a certain extent. The question remains: is the primary location of the ERalpha also the plasma membrane? If that is the case, it will be a challenging task to identify the molecular events that underlie the plasma membrane-to-nucleus movement of ERalpha. The internalization mechanisms for the two 66kDa plasma membrane ERs, following hormone binding, appear to be distinct and without any overlaps. Interestingly, while the major gene regulatory role for ERalpha appears to be at the level of transcription, the nER II has its major functional role in post transcriptional mechanisms. The endoplasmic reticulum associated anchor protein-55 (ap55) that was recently reported from the author's laboratory needs a closer look. It is a high affinity estrogen binding protein that anchors the estrogen receptor activation factor (E-RAF) in an estrogen-mediated event. It will be interesting to examine whether ap55 bears any structural similarity with either ERalpha or ERbeta.

Cell cycle arrest and apoptosis induced by SART-1 gene transduction.

UNLABELLED: The biological function of the SART-1 gene product is demonstrated and its potential as a target for cancer gene therapy is discussed. MATERIALS AND METHODS: The SART-1 gene was transduced by a recombinant adenovirus vector and its expression was promoted by a CMV promoter. RESULTS: The transduction efficiency by recombinant adenoviruses in A549 and MCF-7 cells was determined using a vector expressing luciferase, which showed high expression in the cells. Cell count analysis using Trypan-Blue dye exclusion showed that SART-1 gene transduction inhibited cell growth. Flow cytometry analysis suggested that SART-1 gene transduction induced cell cycle arrest followed by apoptosis. Western blot analysis confirmed that the apoptosis pathway was activated by SART-1 gene transduction. CONCLUSION: These results show that SART-1 gene transduction induces cell cycle arrest leading to apoptosis and suggest the possibility of gene therapy against cancer. In addition, SART-1 is known to be a tumor antigen in a range of cancers recognized by T cells, thus a potential strategy would be the combination of suicide gene therapy with immuno-gene therapy.

LSm proteins form heptameric rings that bind to RNA via repeating motifs.

Members of the LSm family of proteins share the Sm fold--a closed barrel comprising five anti-parallel beta strands with an alpha helix stacked on the top. The fold forms a subunit of hexameric or heptameric rings of approximately 7nm in diameter. Interactions between neighboring subunits center on an anti-parallel interaction of the fourth and fifth beta strands. In the lumen of the ring, the subunits have the same spacing as nucleotides in RNA, enabling the rings to bind to single-stranded RNA via a repeating motif. Eubacteria and archaea build homohexamers and homoheptamers, respectively, whereas eukaryotes use >18 LSm paralogs to build at least six different heteroheptameric rings. The four different rings in the nucleus that permanently bind small nuclear RNAs and function in pre-mRNA maturation are called Sm rings. The two different rings that transiently bind to RNAs and, thereby, assist in the degradation of mRNA in the cytoplasm and the maturation of a wide spectrum of RNAs in the nucleus are called LSm rings.

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor.

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.

The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons.

Alu repetitive elements can be inserted into mature messenger RNAs via a splicing-mediated process termed exonization. To understand the molecular basis and the regulation of the process of turning intronic Alus into new exons, we compiled and analyzed a data set of human exonized Alus. We revealed a mechanism that governs 3' splice-site selection in these exons during alternative splicing. On the basis of these findings, we identified mutations that activated the exonization of a silent intronic Alu.

Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus.

The m(7)G caps of most spliceosomal snRNAs and certain snoRNAs are converted posttranscriptionally to 2,2,7-trimethylguanosine (m(3)G) cap structures. Here, we show that yeast Tgs1p, an evolutionarily conserved protein carrying a signature of S-AdoMet methyltransferase, is essential for hypermethylation of the m(7)G caps of both snRNAs and snoRNAs. Deletion of the yeast TGS1 gene abolishes the conversion of the m(7)G to m(3)G caps and produces a cold-sensitive splicing defect that correlates with the retention of U1 snRNA in the nucleolus. Consistently, Tgs1p is also localized in the nucleolus. Our results suggest a trafficking pathway in which yeast snRNAs and snoRNAs cycle through the nucleolus to undergo m(7)G cap hypermethylation.

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor.

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4-U6 and U6-U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110(nrb) or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.

The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes.

The association of the U4/U6.U5 tri-snRNP with pre-spliceosomes is a poorly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential role in this process. Characterization by cDNA cloning of the 65 and 110 kDa proteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a block in the formation of mature spliceosomes, but had no effect on the integrity of the U4/U6.U5 tri-snRNP. Spliceosome assembly and splicing catalysis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since both proteins contain an N-terminal RS domain, they could mediate the association of the tri-snRNP with pre-spliceosomes by interaction with members of the SR protein family.

snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN.

Splicing snRNPs (small nuclear ribonucleoproteins) are essential sub-units of the spliceosome. Here we report the establishment of stable cell lines expressing fluorescently tagged SmB, a core snRNP protein. Analysis of these stable cell lines has allowed us to characterize the nuclear pathway that leads to snRNP accumulation in nuclear speckles and has identified a limiting nucleolar step in the pathway that can be saturated by overexpression of Sm proteins. After nuclear import, newly assembled snRNPs accumulate first in a subset of Cajal bodies that contain both p80-coilin and the survival of motor neurons protein (SMN) and not in bodies that contain p80-coilin but lack SMN. Treatment of cells with leptomycin B (LMB) inhibits both the accumulation of snRNPs in nuclear bodies and their subsequent accumulation in speckles. The formation of Cajal bodies is enhanced by Sm protein expression and the assembly of new snRNPs. Formation of heterokaryons between HeLa cell lines expressing Sm proteins and primary cells that usually lack Cajal bodies results in the detection of Cajal bodies in primary cell nuclei. Transient over-expression of exogenous SmB alone is sufficient to induce correspondingly transient Cajal body formation in primary cells. These data indicate that the level of snRNP protein expression and snRNP assembly, rather than the expression levels of p80-coilin or SMN, may be a key trigger for Cajal body formation.

Structure and function in the nucleus.

Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.

Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast.

We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose- repressible promoter. After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate. This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2. Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed. These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.