GPATCH4 regulates rRNA and snRNA 2′-O-methylation in both DHX15-dependent and DHX15-independent manners

Abstract Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2′-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2′-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2′-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.

[1]  M. Dermit,et al.  TREX reveals proteins that bind to specific RNA regions in living cells , 2024, Nature methods.

[2]  Qiulian Wu,et al.  LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2’-O-methylation , 2023, Nature communications.

[3]  M. Dermit,et al.  TREX reveals proteins that bind to specific RNA regions in living cells , 2023, bioRxiv.

[4]  M. Graille,et al.  N 2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing , 2023, Nucleic acids research.

[5]  M. Bohnsack,et al.  Molecular functions of RNA helicases during ribosomal subunit assembly , 2023, Biological chemistry.

[6]  U. Kutay,et al.  Ribosome biogenesis factors—from names to functions , 2023, The EMBO journal.

[7]  Yue Huang,et al.  DHX15 is involved in SUGP1-mediated RNA missplicing by mutant SF3B1 in cancer , 2022, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Ficner,et al.  Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1 , 2022, Proceedings of the National Academy of Sciences of the United States of America.

[9]  C. Schneider,et al.  Caught in the act—Visualizing ribonucleases during eukaryotic ribosome assembly , 2022, Wiley interdisciplinary reviews. RNA.

[10]  H. Nielsen,et al.  Ribosomal RNA 2’-O-methylation dynamics impact cell fate decisions , 2022, bioRxiv.

[11]  M. Bohnsack,et al.  Prp43/DHX15 exemplify RNA helicase multifunctionality in the gene expression network , 2022, Nucleic acids research.

[12]  T. Ke,et al.  Identification and characterization of a special type of subnuclear structure: AGGF1‐coated paraspeckles , 2022, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  Marleen Heinrichs,et al.  The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation , 2022, Nature communications.

[14]  M. Ares,et al.  Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling , 2021, bioRxiv.

[15]  F. Dequiedt,et al.  DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity , 2021, Nature Communications.

[16]  P. Ménard,et al.  Regulation of translation by site-specific ribosomal RNA methylation , 2021, Nature Structural & Molecular Biology.

[17]  K. Pan,et al.  The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly , 2021, Nature Communications.

[18]  M. Jurica,et al.  A model for DHX15 mediated disassembly of A-complex spliceosomes , 2021, bioRxiv.

[19]  D. Lafontaine,et al.  Nopp140-chaperoned 2′-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity , 2021, Genes & development.

[20]  M. Okuwaki,et al.  G-patch domain-containing protein 4 localizes to both the nucleoli and Cajal bodies and regulates cell growth and nucleolar structure. , 2021, Biochemical and biophysical research communications.

[21]  H. Nielsen,et al.  RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2′-O-methylation , 2021, Nucleic acids research.

[22]  R. Ficner,et al.  Regulation of DEAH-box RNA helicases by G-patch proteins , 2021, Biological chemistry.

[23]  K. Grønbæk,et al.  Profiling of ribose methylations in ribosomal RNA from diffuse large B-cell lymphoma patients for evaluation of ribosomes as drug targets , 2020, NAR cancer.

[24]  M. Bohnsack,et al.  The DExD box ATPase DDX55 is recruited to domain IV of the 28S ribosomal RNA by its C-terminal region , 2020, RNA biology.

[25]  Joshua A. Riback,et al.  The nucleolus as a multiphase liquid condensate , 2020, Nature Reviews Molecular Cell Biology.

[26]  S. Jonas,et al.  Structural basis for DEAH-helicase activation by G-patch proteins , 2020, Proceedings of the National Academy of Sciences.

[27]  Anthony J. Cesnik,et al.  Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder , 2020, bioRxiv.

[28]  H. Urlaub,et al.  Structural analysis of the intrinsically disordered splicing factor Spp2 and its binding to the DEAH-box ATPase Prp2 , 2020, Proceedings of the National Academy of Sciences.

[29]  A. Bhardwaj,et al.  Nopp140-mediated concentration of telomerase in Cajal bodies regulates telomere length , 2019, Molecular biology of the cell.

[30]  Ling-Ling Chen,et al.  Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus. , 2019, Molecular cell.

[31]  M. Bohnsack,et al.  Uncovering the assembly pathway of human ribosomes and its emerging links to disease , 2019, The EMBO journal.

[32]  H. Nielsen,et al.  Sequencing-based methods for detection and quantitation of ribose methylations in RNA. , 2019, Methods.

[33]  Jianlin Lei,et al.  Structures of the human spliceosomes before and after release of the ligated exon , 2018, Cell Research.

[34]  H. Nielsen,et al.  SIRT7-Dependent Deacetylation of Fibrillarin Controls Histone H2A Methylation and rRNA Synthesis during the Cell Cycle. , 2018, Cell reports.

[35]  J. Woolford,et al.  Ribosome assembly coming into focus , 2018, Nature Reviews Molecular Cell Biology.

[36]  H. Zoghbi,et al.  RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins , 2018, Cell reports.

[37]  C. Plisson-Chastang,et al.  The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor , 2018, PLoS genetics.

[38]  M. Bohnsack,et al.  Modifications in small nuclear RNAs and their roles in spliceosome assembly and function , 2018, Biological chemistry.

[39]  J. Yates,et al.  Spliceosome Profiling Visualizes Operations of a Dynamic RNP at Nucleotide Resolution , 2018, Cell.

[40]  B. Klaholz,et al.  Visualization of chemical modifications in the human 80S ribosome structure , 2017, Nature.

[41]  Henrik Nielsen,et al.  Substoichiometric ribose methylations in spliceosomal snRNAs. , 2017, Organic & biomolecular chemistry.

[42]  Lin He,et al.  Identification of a 35S U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP) complex intermediate in spliceosome assembly , 2017, The Journal of Biological Chemistry.

[43]  V. Cowling,et al.  DHX15 regulates CMTR1-dependent gene expression and cell proliferation , 2017, Life Science Alliance.

[44]  M. Bohnsack,et al.  The G-patch protein NF-κB-repressing factor mediates the recruitment of the exonuclease XRN2 and activation of the RNA helicase DHX15 in human ribosome biogenesis , 2017, Nucleic acids research.

[45]  R. Lührmann,et al.  Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2 , 2016, Nucleic acids research.

[46]  K. Entian,et al.  Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function , 2016, RNA biology.

[47]  D. Görlich,et al.  Effects of the Bowen-Conradi syndrome mutation in EMG1 on its nuclear import, stability and nucleolar recruitment , 2016, Human molecular genetics.

[48]  S. Goldman,et al.  The human box C/D snoRNAs U3 and U8 are required for pre-rRNA processing and tumorigenesis , 2016, Oncotarget.

[49]  Anders H. Lund,et al.  Profiling of 2′-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity , 2016, Nucleic acids research.

[50]  D. Sturgill,et al.  Cajal bodies are linked to genome conformation , 2016, Nature Communications.

[51]  H. Urlaub,et al.  Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways , 2016, RNA biology.

[52]  E. Hurt,et al.  The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation , 2016, Nucleic acids research.

[53]  F. You,et al.  Nlrp6 regulates intestinal antiviral innate immunity , 2015, Science.

[54]  S. Richard,et al.  Emerging Roles of Disordered Sequences in RNA-Binding Proteins. , 2015, TIBS -Trends in Biochemical Sciences. Regular ed.

[55]  B. Klaholz,et al.  Structure of the human 80S ribosome , 2015, Nature.

[56]  K. Entian,et al.  Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1 , 2015, Nucleic acids research.

[57]  I. Ebersberger,et al.  The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21 , 2014, Nucleic acids research.

[58]  Jan Gorodkin,et al.  Profiling of ribose methylations in RNA by high-throughput sequencing. , 2014, Angewandte Chemie.

[59]  Oliver Kohlbacher,et al.  Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins , 2014, Nature Methods.

[60]  Y. Henry,et al.  The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis , 2014, Nucleic acids research.

[61]  H. Ichijo,et al.  The DEAH-Box RNA Helicase DHX15 Activates NF-κB and MAPK Signaling Downstream of MAVS During Antiviral Responses , 2014, Science Signaling.

[62]  A. Gregory Matera,et al.  A day in the life of the spliceosome , 2014, Nature Reviews Molecular Cell Biology.

[63]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[64]  Xin Sheng Zhao,et al.  Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation , 2012, Nucleic acids research.

[65]  M. Bohnsack,et al.  The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA , 2012, Wiley interdisciplinary reviews. RNA.

[66]  Zhaoyang Niu,et al.  Tumor suppressor RBM5 directly interacts with the DExD/H‐box protein DHX15 and stimulates its helicase activity , 2012, FEBS letters.

[67]  U. Fischer,et al.  Biogenesis of spliceosomal small nuclear ribonucleoproteins , 2011, Wiley interdisciplinary reviews. RNA.

[68]  J. Gallagher,et al.  The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing , 2011, Nucleic acids research.

[69]  David Tollervey,et al.  Prp43 Bound at Different Sites on the Pre-rRNA Performs Distinct Functions in Ribosome Synthesis , 2009, Molecular cell.

[70]  Yusuke Nakamura,et al.  Involvement of G‐patch domain containing 2 overexpression in breast carcinogenesis , 2009, Cancer science.

[71]  David Tollervey,et al.  Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs , 2009, Proceedings of the National Academy of Sciences.

[72]  M. Paine,et al.  TFIP11 Interacts with mDEAH9, an RNA Helicase Involved in Spliceosome Disassembly , 2008, International journal of molecular sciences.

[73]  A. Blomberg,et al.  Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping. , 2008, RNA.

[74]  Chia-Yi Hsu,et al.  Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription , 2008, Journal of biomedical science.

[75]  T. Hughes,et al.  Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. , 2007, Genes & development.

[76]  W. Heyer,et al.  NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. , 2003, Analytical biochemistry.

[77]  H. Hauser,et al.  Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[78]  Xiao Zhen Zhou,et al.  The Pin2/TRF1-Interacting Protein PinX1 Is a Potent Telomerase Inhibitor , 2001, Cell.

[79]  C. Pai,et al.  Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization , 1999, Molecular and Cellular Biology.

[80]  H. Hauser,et al.  Constitutive silencing of IFN‐β promoter is mediated by NRF (NF‐κB‐repressing factor), a nuclear inhibitor of NF‐κB , 1999 .

[81]  J. Warner,et al.  The economics of ribosome biosynthesis in yeast. , 1999, Trends in biochemical sciences.

[82]  E. Koonin,et al.  G-patch: a new conserved domain in eukaryotic RNA-processing proteins and type D retroviral polyproteins. , 1999, Trends in biochemical sciences.

[83]  J. Abelson,et al.  Prp43: An RNA helicase-like factor involved in spliceosome disassembly. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[84]  B. Peculis,et al.  The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation , 1997, Molecular and cellular biology.

[85]  Tamás Kiss,et al.  Site-Specific Pseudouridine Formation in Preribosomal RNA Is Guided by Small Nucleolar RNAs , 1997, Cell.

[86]  J. Steitz,et al.  A new method for detecting sites of 2'-O-methylation in RNA molecules. , 1997, RNA.

[87]  Tamás Kiss,et al.  Site-Specific Ribose Methylation of Preribosomal RNA: A Novel Function for Small Nucleolar RNAs , 1996, Cell.

[88]  G. Blobel,et al.  Nopp 140 shuttles on tracks between nucleolus and cytoplasm , 1992, Cell.

[89]  J. Steitz,et al.  A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells , 1988, The Journal of cell biology.

[90]  R. Maser,et al.  U6 small nuclear RNA is transcribed by RNA polymerase III. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[91]  M. Bohnsack,et al.  Crosslinking Methods to Identify RNA Methyltransferase Targets In Vivo. , 2017, Methods in molecular biology.

[92]  D. Tollervey,et al.  Identification of RNA helicase target sites by UV cross-linking and analysis of cDNA. , 2012, Methods in enzymology.