Figure 6. NHL-2 and CGH-1 Act after miRNA Biogenesis to Promote miRNA-Mediated Gene Regulation in Association with the Core miRISC(A) miRNA northern analysis of total RNA extracted from wild-type,
nhl-2(0), negative control RNAi,
cgh-1 RNAi, or an
alg-1(0) mutant. Only mutations of
alg-1, encoding the miRNA-associated Argonaute and core miRISC component, lead to defects in miRNA biogenesis (misprocessing and underaccumulation of mature miRNAs).(B) Immunoprecipitation of GFP::NHL-2 from extracts of larvae coprecipitates ALG-1 and ALG-2.(C) Immunoprecipitation of CGH-1 coprecipitates ALG-1/2, AIN-1,and NHL-2. RNase treatment of immunoprecipitated material reduces CGH-1 binding to core miRISC components but does not dramatically affect its interaction with NHL-2.(D) Coimmunoprecipitation of ALG-1/2 and AIN-1 by CGH-1 antiserum is not affected in the absence of
nhl-2.(E) A model representing potential roles for NHL-2 in the posttranscriptional regulation of miRNA targets. We suggest that miRISC has a basal level of repressive activity in the absence of NHL-2, leading to the reduction of protein products derived from miRNA target transcripts. Our models suggest that NHL-2 augments this basal level of activity by physically associating with miRISC. Proposed points of function and potential activities of NHL-2 include (1) an enhancement of CGH-1:miRISC:target interaction by increasing the stability or fidelity of miRNA:target interactions; (2) a stimulatory role for NHL-2 in modulating intrinsic activity of CGH-1 and/or miRISC components; and (3) a role for NHL-2 independent of CGH-1 or other known miRISC components.