HRPK-1, a conserved KH-domain protein, modulates microRNA activity during Caenorhabditis elegans development

Autoři: Li Li aff001;  Isana Veksler-Lublinsky aff002;  Anna Zinovyeva aff001
Působiště autorů: Division of Biology, Kansas State University, Manhattan, Kansas, United States of America aff001;  Department of Software and Information Systems Engineering, Ben-Gurion University, Beer-sheva, Israel aff002
Vyšlo v časopise: HRPK-1, a conserved KH-domain protein, modulates microRNA activity during Caenorhabditis elegans development. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008067
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008067


microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity.

Klíčová slova:

Alleles – Caenorhabditis elegans – Embryos – Gene expression – Larvae – MicroRNAs – Phenotypes – RNA-binding proteins


1. Rajman M, Schratt G. MicroRNAs in neural development: from master regulators to fine-tuners. Development. Oxford University Press for The Company of Biologists Limited; 2017 Jul 1;144(13):2310–22. doi: 10.1242/dev.144337 28676566

2. Ivey KN, Srivastava D. microRNAs as Developmental Regulators. Cold Spring Harb Perspect Biol. Cold Spring Harbor Lab; 2015 Jul 1;7(7):a008144. doi: 10.1101/cshperspect.a008144 26134312

3. Bartel DP. Metazoan MicroRNAs. Cell. 2018 Mar 22;173(1):20–51. doi: 10.1016/j.cell.2018.03.006 29570994

4. Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing. Nature Publishing Group. Nature Publishing Group; 2015 Jul;16(7):421–33.

5. Treiber T, Treiber N, Plessmann U, Harlander S, Daiß J-L, Eichner N, et al. A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis. Molecular Cell. 2017 Apr 20;66(2):270–284.e13. doi: 10.1016/j.molcel.2017.03.014 28431233

6. Schopp IM, Amaya Ramirez CC, Debeljak J, Kreibich E, Skribbe M, Wild K, et al. Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat Comms. Nature Publishing Group; 2017 Jun 6;8:15690.

7. Hammell CM, Lubin I, Boag PR, Blackwell TK, Ambros V. nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell. 2009 Mar 6;136(5):926–38. doi: 10.1016/j.cell.2009.01.053 19269369

8. HOck J, Weinmann L, Ender C, Rüdel S, Kremmer E, Raabe M, et al. Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells. EMBO Rep. EMBO Press; 2007 Nov;8(11):1052–60. doi: 10.1038/sj.embor.7401088 17932509

9. Alessi AF, Khivansara V, Han T, Freeberg MA, Moresco JJ, Tu PG, et al. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1. Proc Natl Acad Sci USA. National Academy of Sciences; 2015 Dec 29;112(52):E7213–22. doi: 10.1073/pnas.1509499112 26669440

10. Parry DH, Xu J, Ruvkun G. A Whole-Genome RNAi Screen for C. elegans miRNA Pathway Genes. Current Biology. 2007 Dec;17(23):2013–22. doi: 10.1016/j.cub.2007.10.058 18023351

11. Nolde MJ, Saka N, Reinert KL, Slack FJ. The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1. Dev Biol. 2007 May 15;305(2):551–63. doi: 10.1016/j.ydbio.2007.02.040 17412319

12. Ding XC, Slack FJ, Großhans H. The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation. Cell Cycle. 2008 Oct;7(19):3083–90. doi: 10.4161/cc.7.19.6778 18818519

13. Rausch M, Ecsedi M, Bartake H, Müllner A, Großhans H. A genetic interactome of the let-7 microRNA in C. elegans. Dev Biol. 2015 May 15;401(2):276–86. doi: 10.1016/j.ydbio.2015.02.013 25732775

14. Zinovyeva AY, Veksler-Lublinsky I, Vashisht AA, Wohlschlegel JA, Ambros VR. Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. Proc Natl Acad Sci USA. 2015 Sep 22;112(38):E5271–80. doi: 10.1073/pnas.1506576112 26351692

15. Geuens T, Bouhy D, Timmerman V. The hnRNP family: insights into their role in health and disease. Hum Genet. Springer Berlin Heidelberg; 2016 Aug;135(8):851–67. doi: 10.1007/s00439-016-1683-5 27215579

16. Dreyfuss G, Kim VN, Kataoka N. Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol. Nature Publishing Group; 2002 Mar;3(3):195–205. doi: 10.1038/nrm760 11994740

17. Kooshapur H, Choudhury NR, Simon B, Mühlbauer M, Jussupow A, Fernandez N, et al. Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing by hnRNP A1. Nat Comms. Nature Publishing Group; 2018 Jun 26;9(1):2479.

18. Svitkin YV, Yanagiya A, Karetnikov AE, Alain T, Fabian MR, Khoutorsky A, et al. Control of translation and miRNA-dependent repression by a novel poly(A) binding protein, hnRNP-Q. Lykke-Andersen J, editor. PLoS Biol. Public Library of Science; 2013;11(5):e1001564. doi: 10.1371/journal.pbio.1001564 23700384

19. Siomi H, Matunis MJ, Michael WM, Dreyfuss G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Research. Oxford University Press; 1993 Mar 11;21(5):1193–8. doi: 10.1093/nar/21.5.1193 8464704

20. Valverde R, Edwards L, Regan L. Structure and function of KH domains. FEBS J. Blackwell Publishing Ltd; 2008 Jun;275(11):2712–26. doi: 10.1111/j.1742-4658.2008.06411.x 18422648

21. Musco G, Stier G, Joseph C, Castiglione Morelli MA, Nilges M, Gibson TJ, et al. Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. Cell. 1996 Apr 19;85(2):237–45. doi: 10.1016/s0092-8674(00)81100-9 8612276

22. Kruse C, Willkomm D, Gebken J, Schuh A, Stossberg H, Vollbrandt T, et al. The multi-KH protein vigilin associates with free and membrane-bound ribosomes. Cell Mol Life Sci. Birkhäuser-Verlag; 2003 Oct;60(10):2219–27. doi: 10.1007/s00018-003-3235-0 14618268

23. García-Mayoral MF, Hollingworth D, Masino L, Díaz-Moreno I, Kelly G, Gherzi R, et al. The structure of the C-terminal KH domains of KSRP reveals a noncanonical motif important for mRNA degradation. Structure. 2007 Apr;15(4):485–98. doi: 10.1016/j.str.2007.03.006 17437720

24. Zabinsky RA, Weum BM, Cui M, Han M. RNA Binding Protein Vigilin Collaborates with miRNAs To Regulate Gene Expression for Caenorhabditis elegans Larval Development. G3: Genes|Genomes|Genetics. G3: Genes, Genomes, Genetics; 2017 Aug 7;7(8):2511–8.

25. Shin CH, Lee H, Kim HR, Choi KH, Joung J-G, Kim HH. Regulation of PLK1 through competition between hnRNPK, miR-149-3p and miR-193b-5p. Cell Death Differ. Nature Publishing Group; 2017 Nov;24(11):1861–71. doi: 10.1038/cdd.2017.106 28708135

26. Fan B, Sutandy FXR, Syu G-D, Middleton S, Yi G, Lu K-Y, et al. Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication. Molecular & Cellular Proteomics. American Society for Biochemistry and Molecular Biology; 2015 Nov;14(11):2878–86.

27. Fan B, Lu K-Y, Reymond Sutandy FX, Chen Y-W, Konan K, Zhu H, et al. A human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) recognizes the 5' terminal sequence of the hepatitis C virus RNA. Molecular & Cellular Proteomics. 2014 Jan;13(1):84–92.

28. Akay A, Craig A, Lehrbach N, Larance M, Pourkarimi E, Wright JE, et al. RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans. Open Biology. Royal Society Journals; 2013 Nov 1;3(11):130151. doi: 10.1098/rsob.130151 24258276

29. Ambros V, Ruvkun G. Recent Molecular Genetic Explorations of Caenorhabditis elegans MicroRNAs. Genetics. 2018 Jul;209(3):651–73. doi: 10.1534/genetics.118.300291 29967059

30. Abbott AL, Alvarez-Saavedra E, Miska EA, Lau NC, Bartel DP, Horvitz HR, et al. The let-7 MicroRNA Family Members mir-48, mir-84, and mir-241 Function Together to Regulate Developmental Timing in Caenorhabditis elegans. Developmental Cell. 2005 Sep;9(3):403–14. doi: 10.1016/j.devcel.2005.07.009 16139228

31. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000 Feb 24;403(6772):901–6. doi: 10.1038/35002607 10706289

32. Abrahante JE, Miller EA, Rougvie AE. Identification of heterochronic mutants in Caenorhabditis elegans: temporal misexpression of a collagen:: green fluorescent protein fusion gene. Genetics. Genetics Soc America; 1998;149(3):1335–51. 9649524

33. Vella MC, Choi E-Y, Lin S-Y, Reinert K, Slack FJ. The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. Genes & Development. Cold Spring Harbor Lab; 2004 Jan 15;18(2):132–7.

34. Johnston RJ, Hobert O. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature. 2003 Dec 18;426(6968):845–9. doi: 10.1038/nature02255 14685240

35. Chang SS, Johnston RJR, Hobert OO. A transcriptional regulatory cascade that controls left/right asymmetry in chemosensory neurons of C. elegans. Genes & Development. 2003 Sep 1;17(17):2123–37.

36. Sarin S, O'Meara MM, Flowers EB, Antonio C, Poole RJ, Didiano D, et al. Genetic Screens for Caenorhabditis elegans Mutants Defective in Left/Right Asymmetric Neuronal Fate Specification. Genetics. 2007 Aug 1;176(4):2109–30. doi: 10.1534/genetics.107.075648 17717195

37. Alvarez-Saavedra E, Horvitz HR. Many families of C. elegans microRNAs are not essential for development or viability. Curr Biol. 2010 Feb 23;20(4):367–73. doi: 10.1016/j.cub.2009.12.051 20096582

38. McJunkin K, Ambros V. The embryonic mir-35 family of microRNAs promotes multiple aspects of fecundity in Caenorhabditis elegans. G3: Genes|Genomes|Genetics. 2014 Sep;4(9):1747–54.

39. Reinke AW, Mak R, Troemel ER, Bennett EJ. In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans. Sci Adv. 2017 May;3(5):e1602426. doi: 10.1126/sciadv.1602426 28508060

40. Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. Cold Spring Harbor Lab; 2008 Dec;14(12):2580–96. doi: 10.1261/rna.1351608 18978028

41. Dallaire A, Frédérick P-M, Simard MJ. Somatic and Germline MicroRNAs Form Distinct Silencing Complexes to Regulate Their Target mRNAs Differently. Developmental Cell. 2018 Oct 22;47(2):239–247.e4. doi: 10.1016/j.devcel.2018.08.022 30245155

42. Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. 2008 Dec;14(12):2580–96. doi: 10.1261/rna.1351608 18978028

43. Brenner S. The Genetics of CAENORHABDITIS ELEGANS. Genetics. 1974 May 7;77:71–94. 4366476

44. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003 Jan 16;421(6920):231–7. doi: 10.1038/nature01278 12529635

45. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics. Genetics; 2015 Aug;200(4):1035–49. doi: 10.1534/genetics.115.178335 26044593

46. Mello CC, Kramer JM, Stinchcomb D, Ambros V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. European Molecular Biology Organization; 1991 Dec;10(12):3959–70. 1935914

47. Zinovyeva AY, Bouasker S, Simard MJ, Hammell CM, Ambros V. Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression. Public Library of Science; 2014 Apr;10(4):e1004286.

48. Zou Y, Chiu H, Zinovyeva A, Ambros V, Chuang C-F, Chang C. Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers. Science. 2013 Apr 19;340(6130):372–6. doi: 10.1126/science.1231321 23599497

49. Miller BR, Wei T, Fields CJ, Sheng P, Xie M. Near-infrared fluorescent northern blot. RNA. Cold Spring Harbor Lab; 2018 Dec;24(12):1871–7. doi: 10.1261/rna.068213.118 30201850

50. Gu W, Claycomb J, Batista P, Mello C, Conte D. Cloning Argonaute-Associated Small RNAs from Caenorhabditis elegans. In: Hobman TC, Duchaine TF, editors. Methods in Molecular Biology. Humana Press; 2011. pp. 251–280–280.

51. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biology. BioMed Central; 2010;11(10):R106–12. doi: 10.1186/gb-2010-11-10-r106 20979621

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