Abstract
Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher K(cat) and lower K(m) values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates.
Highlights
Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes
Dawdle has been proposed to participate in miRNA biogenesis by facilitating DCL1 access to pri-miRNA (Yu et al, 2008), while CBP20 and CBP80 may mediate the interaction between the nuclear Cap-binding complex and the miRNA processing machinery (Laubinger et al, 2008; Yu et al, 2008)
Deep sequencing analysis of miRNA levels in vivo confirm that they are substantially elevated in the dcl1/hyl1-2 plants compared with hyl1-2 plants
Summary
Dicer-Like (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. Dicer-Like (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. Dcl, which harbors a T-DNA insertion in the last dsRNA-binding domain, and dcl, which has a point mutation in the catalytic site of the RNAseIIIb domain, accumulate reduced levels of miRNAs (Kurihara and Watanabe, 2004; Kurihara et al, 2006; Willmann et al, 2011) Another allele with a lesion in the helicase domain, dcl, was identified as a semidominant suppressor of hyl (Tagami et al, 2009). It promotes miRNA processing in the absence of HYL1, but impairs miRNA processing in the presence of HYL1 (Tagami et al, 2009)
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