Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

Anna Y. Zinovyeva; Isana Veksler-Lublinsky; Ajay A. Vashisht; James A. Wohlschlegel; Victor R. Ambros; H. Robert Horvitz

doi:10.1073/pnas.1506576112

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

Anna Y. Zinovyeva, Isana Veksler-Lublinsky, Ajay A. Vashisht, James A. Wohlschlegel, Victor R. Ambros, H. Robert Horvitz

Research output: Contribution to journal › Article › peer-review

Abstract

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans micro-RNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA^∗ (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA^∗ strands, with some microRNA^∗ strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA^∗ strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA^∗ strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA^∗ strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA^∗ ejection steps of miRISC maturation.

Original language	American English
Pages (from-to)	E5271-E5280
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	112
Issue number	38
DOIs	https://doi.org/10.1073/pnas.1506576112
State	Published - 22 Sep 2015
Externally published	Yes

Keywords

ALG-1
Argonaute
MicroRNA
MicroRNA
Passenger

All Science Journal Classification (ASJC) codes

General

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10.1073/pnas.1506576112

Cite this

Zinovyeva, A. Y., Veksler-Lublinsky, I., Vashisht, A. A., Wohlschlegel, J. A., Ambros, V. R., & Horvitz, H. R. (2015). Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. Proceedings of the National Academy of Sciences of the United States of America, 112(38), E5271-E5280. https://doi.org/10.1073/pnas.1506576112

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title = "Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal",

abstract = "MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans micro-RNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA∗ (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA∗ strands, with some microRNA∗ strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA∗ strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA∗ strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA∗ strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA∗ ejection steps of miRISC maturation.",

keywords = "ALG-1, Argonaute, MicroRNA, MicroRNA, Passenger",

author = "Zinovyeva, {Anna Y.} and Isana Veksler-Lublinsky and Vashisht, {Ajay A.} and Wohlschlegel, {James A.} and Ambros, {Victor R.} and Horvitz, {H. Robert}",

year = "2015",

month = sep,

day = "22",

doi = "10.1073/pnas.1506576112",

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Zinovyeva, AY, Veksler-Lublinsky, I, Vashisht, AA, Wohlschlegel, JA, Ambros, VR & Horvitz, HR 2015, 'Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal', Proceedings of the National Academy of Sciences of the United States of America, vol. 112, no. 38, pp. E5271-E5280. https://doi.org/10.1073/pnas.1506576112

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. / Zinovyeva, Anna Y.; Veksler-Lublinsky, Isana; Vashisht, Ajay A. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 38, 22.09.2015, p. E5271-E5280.

Research output: Contribution to journal › Article › peer-review

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T1 - Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

AU - Zinovyeva, Anna Y.

AU - Veksler-Lublinsky, Isana

AU - Vashisht, Ajay A.

AU - Wohlschlegel, James A.

AU - Ambros, Victor R.

AU - Horvitz, H. Robert

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AB - MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans micro-RNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA∗ (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA∗ strands, with some microRNA∗ strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA∗ strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA∗ strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA∗ strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA∗ ejection steps of miRISC maturation.

KW - ALG-1

KW - Argonaute

KW - MicroRNA

KW - Passenger

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Zinovyeva AY, Veksler-Lublinsky I, Vashisht AA, Wohlschlegel JA, Ambros VR, Horvitz HR. Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. Proceedings of the National Academy of Sciences of the United States of America. 2015 Sep 22;112(38):E5271-E5280. doi: 10.1073/pnas.1506576112

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

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