NHL-2 Influences 22G RNA biogenesis to Mediate Germline Chromosomal Integrity. — University of Technology
  1. Schedule
  2. Young Investigator
  3. NHL-2 Influences 22G RNA biogenesis to Mediate Germline Chromosomal Integrity.

NHL-2 Influences 22G RNA biogenesis to Mediate Germline Chromosomal Integrity. (14253)

Gregory M Davis 1 , Zhiping Weng 2 , Wai Y Low , Julie M Claycomb 3 , Peter R Boag 1
  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  2. Biochemistry and Molecular Pharmacology,, University of Massachusetts Medical School, Worcester, MA, USA
  3. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

Background: The TRIM-NHL protein, NHL-2, functions as a miRNA RISC co-factor in the somatic tissues of Caenorhabditis elegans, where it is required for the efficacy of   let-7 miRNA function.  NHL-2 is also highly expressed in the germline, however, no evidence to date has shown that miRNAs contribute significantly to germline function. Preliminary analysis of nhl-2 null mutants showed multiple germline defects, including abnormal oocyte chromosome organisation, reduced brood size and embryonic lethality. This suggests that NHL-2 is required for germline function, however, a mechanistic understanding remains unknown.

Aims: To identify germline specific pathways that require NHL-2 for optimal germline function, including chromosomal maintenance.

Methods: To investigate potential pathways that require NHL-2, we conducted an RNAi screen in which 11,955 genes (55% of the C. elegans genome) were knocked down in wild-type and nhl-2 null mutant worms. Gene expression analysis used a combination of deep sequencing and qRT-PCR analysis. Analysis of interacting proteins used a combination of immunoprecipitation, mass spectrometry and western blotting. Cell biology was achieved by using an immunohistochemistry approach.

Results: Our RNAi screen identified 40 genes, including the DEAD-box helicase, drh-3, which is required for the biogenesis of germline-specific small RNAs known as 22G RNAs.  This class of small RNAs are required for germline chromosomal integrity and guard the genome against transposons, pseudogenes and cryptic loci. Knockdown of drh-3 in nhl-2 mutants leads to marked chromosomal abnormalities in oocytes and significantly enhanced embryonic lethality.  Similar phenotypes were observed when other 22G RNA pathway co-factors were knocked down in nhl-2 mutants, including the Argonaut protein CSR-1.  CSR-1 binds 22G small RNAs regulates gene expression and chromosome segregation and co-immunoprecipitates with NHL-2.  Deep sequencing of small RNAs from wild-type and nhl-2 null mutant worms found that the 22G class of small RNAs were significantly depleted in nhl-2 null mutants, while other small RNA classes were unchanged.

Conclusion: Taken together, our results suggest that NHL-2 contributes to genome maintenance and germline chromosomal integrity by influencing the biogenesis of 22G RNAs.

#jajRNA14