Published papers

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E prerRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle. Knockdown of PARN or exogenous expression of an enzyme-dead PARN mutant (D28A) accumulated 18S-E in both the cytoplasm and nucleus. Moreover, expression of D28A accumulated 18S-E in Bystin-associated pre-40S particles, suggesting that the enzymatic activity of PARN is necessary for the release of 18S-E from Bystin-associated pre-40S particles. Finally, RNase H-based fragmentation analysis and 3'-sequence analysis of 18S-E species present in cells expressing wild-type PARN or D28A suggested that PARN degrades the extended regions encompassing nucleotides 5-44 at the 3' end of mature 18S rRNA. Our results reveal a novel role for PARN in ribosome biogenesis in human cells.

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S preribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006148, 1-23, 2011.

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43-related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention.

Collaborator of alternative reading frame protein (CARF) associates directly with ARF, p53, and/or human double minute 2 protein (HDM2), a ubiquitin-protein ligase, without cofactors and regulates cell proliferation by forming a negative feedback loop. Although ARF, p53, and HDM2 also participate in the regulation of ribosome biogenesis, the involvement of CARF in this process remains unexplored. In this study, we demonstrate that CARF associates with 5'-3' exoribonuclease 2 (XRN2), which plays a major role in both the maturation of rRNA and the degradation of a variety of discarded pre-rRNA species. We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus. This was also observed upon XRN2 knockdown. Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis. These observations suggest that CARF regulates early steps of pre-rRNA processing during ribosome biogenesis by controlling spatial distribution of XRN2 between the nucleoplasm and nucleolus.

SMN (survival motor neuron protein) complexes are essential for the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). During the biogenesis, the SMN complexes bound to UsnRNPs are transported from the cytoplasm to the nucleus, and moved to Cajal body (bodies)/Gems (Cajal/Gems) where the SMN complexes- UsnRNPs are subjected to additional chemical modifications and dissociated to the SMN complexes and the mature UsnRNPs. Although the mature UsnRNPs are assembled into spliceosome with newly transcribed pre-mRNA in the perichromatin fibrils at the chromatin, the role of the dissociated nuclear SMN complexes remains undetermined. In this study, we identified Friend of Prmt1 (FOP chromatin target of Prmt1, CHTOP C1orf77) as a novel component of the nuclear SMN complexes by the biotin affinity purification, coupled with the mass spectrometry-based protein identification. FOP was associated with SMN, Gemines 2, 3, 4, 6, and 8, unrip, and fragile X mental retardation 1 protein (FMR1), as well as with U5and U6 snRNAs in the nucleus, but not with Sm proteins, Gemin5, coilin, and U1 and U2snRNAs. Using the quantitative proteomic method with SILAC coupled with RNA interference, we also showed that FOP is required for the association of the SMN complexes with hnRNPs, histone proteins, and various RNA-binding proteins. It is reported that FOP localizes mainly in the nuclear speckles, binds chromatin, and plays a role in mRNA transcriptional regulation. Our present data suggest that the nuclear SMN complex containing FOP participates in the process of mRNA post-transcriptional regulation. © 2013 Izumikawa K, et al.

Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly-1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc-finger DNA-binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre-rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre-rRNAs. In addition, we show that over-expression of LYAR increases cell proliferation without affecting the expression of c-Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.

During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S.

The U1 small nuclear ribonucleoprotein (snRNP) plays pivotal roles in pre-mRNA splicing and in regulating mRNA length and isoform expression; however, the mechanism of U1 snRNA quality control remains undetermined. Here, we describe a novel surveillance pathway for U1 snRNP biogenesis. Mass spectrometry-based RNA analysis showed that a small population of SMN complexes contains truncated forms of U1 snRNA (U1-tfs) lacking the Sm-binding site and stem loop 4 but containing a 7-monomethylguanosine 5' cap and a methylated first adenosine base. U1-tfs form a unique SMN complex, are shunted to processing bodies and have a turnover rate faster than that of mature U1 snRNA. U1-tfs are formed partly from the transcripts of U1 genes and partly from those lacking the 3' box elements or having defective SL4 coding regions. We propose that U1 snRNP biogenesis is under strict quality control: U1 transcripts are surveyed at the 3'-terminal region and U1-tfs are diverted from the normal U1 snRNP biogenesis pathway.

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage. Molecular & Cellular Proteomics 8: 1552-1565, 2009.

During T cell activation, mitochondrial biogenesis and cellular metabolism are altered to meet the elevated energy demands of protein synthesis, rapid proliferation, and effector T cell function. The mechanisms coupling mitochondrial dynamics to T cell status are unclear. Here, we report that RNA cap methyltransferase 1 (CMTR1) is induced in activated T cells, methylating the first nucleotide on mRNA and U2 small nuclear RNA (snRNA), a component of the spliceosome. Using transcriptomic analyses, we identify a functional splicing module regulating mitochondrial dynamics in T cells, which alters the isoforms of proteins controlling mitochondrial fission and fusion. Through epitranscriptomic control of U2 snRNA and splicing, CMTR1 directs protein isoform selection during T cell activation to promote the development of longer mitochondria with increased respiratory capacity. Thus, CMTR1 upregulation supports the energetic demands of T cell activation, survival, and immune responses.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.