RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

Jacqueline Baca Legendre,Zachary T Campbell,Peggy Kroll-Conner,Phil Anderson,Judith Kimble,Marvin Wickens

doi:10.1074/jbc.m112.397349

Jacqueline Baca Legendre, Zachary T Campbell + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m112.397349

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Abstract

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

Highlights

We purified recombinant versions of STAU-1 mutant proteins, each with a single doublestranded RNA-binding domains (dsRBDs) deleted. (⌬dsRBD2 protein was prone to aggregation and was not pursued.) Each mutant protein bound DS3 doublestranded RNA (dsRNA) (Fig. 2G). ⌬dsRBD3, -4, and -5 proteins bound with similar affinity as wild-type; only ⌬dsRBD1 had a weaker affinity for the dsRNA, implying that it might contribute preferentially to RNA binding (Fig. 2G)
The stau-1 alleles caused increased larval lethality after lir-1(RNAi) compared with wild-type but less than eri-1 (wild-type ϭ 9%, stau-1(⌬dsRBD4) ϭ 49%, stau1(⌬dsRBD2) ϭ 37%, and eri-1(mg366) ϭ 81% larval lethality; Fig. 6). We found that both stau-1 mutant alleles are more sensitive to dpy-13(RNAi) and lir-1(RNAi) compared with wild-type. (The strains tested did not contain mut-16 mutations, which are present in many “wild-type” C. elegans backgrounds and cause alterations in RNA interference (RNAi) sensitivity [43].) These results suggest that mutations in stau-1 enhance RNAi effectiveness and are considered Enhancer of RNAi (Eri)
We describe the biochemical and genetic characteristics of C. elegans STAU-1

Summary

Background

Mutants lacking a single RNA-binding domain enhance RNAi. Conclusion: Staufen associates with target RNAs in vivo. The identity of Staufen mRNA targets in dendrites is not clear, but immunoprecipitation coupled with microarray analysis (RIP-Chip) identified candidate Staufen targets in cultured mammalian cells In these experiments, STAU1 and STAU2 proteins were overexpressed in HEK293T cells, and thousands of transcripts were found to be associated with each protein [42]. Mutant proteins lacking single dsRBDs that are otherwise full-length, still bind well, implying that no single domain is required for high affinity RNA interactions ( dsRBD2 could not be tested). Using STAU-1-specific peptide antibodies and RIP-Chip, we identify mRNA targets of endogenous STAU-1 protein These STAU-1associated mRNAs are diverse in function and show modest overlap with those identified in cultured mammalian cells. Stau-1 genetic mutants display enhanced RNAi phenotypes after exposure to dsRNA, and stau-1;eri-1 double mutants have synthetic germ line defects that cause partial sterility

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