Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processing

Y Iwata,N V Fedoroff,M Takahashi,S M Hamdan

doi:10.1093/nar/gkt667

Abstract

Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.

Highlights

MicroRNAs are small RNAs that regulate gene expression in eukaryotic cells, playing important regulatory roles in many biological processes [1]. miRNAs are transcribed by RNA polymerase II as a precursor RNA, termed the primary miRNA, which contains a hairpin structure with number of bulges and that is capped and polyadenylated at the 50 and 30 ends, respectively
The Nterminal domain of SE appears to be required for RNA binding, the ability of the N-terminally truncated SE protein to stimulate DICER-LIKE 1 (DCL1) activity suggests that it does so directly and that RNA binding is not required for this stimulation
In the present study using highly purified proteins, we find that SE substantially enhances processing of the primiRNA under optimized reaction conditions

Summary

Introduction

MicroRNAs (miRNAs) are small RNAs that regulate gene expression in eukaryotic cells, playing important regulatory roles in many biological processes [1]. miRNAs are transcribed by RNA polymerase II as a precursor RNA, termed the primary miRNA (pri-miRNA), which contains a hairpin structure with number of bulges and that is capped and polyadenylated at the 50 and 30 ends, respectively. Pri-miRNA is processed by an RNaseIII family enzyme to release the miRNA/miRNA* duplex located within the hairpin. Pre-miRNA is cleaved to release the miRNA/miRNA* duplex This is in contrast to animals in which the two steps are mediated by two different RNAseIII-family enzymes, Drosha and Dicer, and take place in the nucleus and the cytoplasm, respectively [2]. Several lines of evidence from Arabidopsis thaliana suggest that a number of proteins act together with DCL1 to ensure efficient and precise miRNA biogenesis. These include HYPONASTIC LEAVES 1 (HYL1), a double-stranded RNA-binding protein (dsRBD) containing two dsRBDs, and SERRATE (SE), a zinc-finger (ZnF) domain-containing protein.

Methods

Results

Conclusion