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Dissecting the Differential Functions of the Two Isoforms of the Essential Argonaute CSR-1 Throughout Caenorhabditis elegans Development.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Dissecting the Differential Functions of the Two Isoforms of the Essential Argonaute CSR-1 Throughout Caenorhabditis elegans Development./
Author:	Charlesworth, Amanda Gene.
Description:	1 online resource (218 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 85-05, Section: B.
Contained By:	Dissertations Abstracts International85-05B.
Subject:	Molecular biology. -
Online resource:	click for full text (PQDT)
ISBN:	9798380841375

Dissecting the Differential Functions of the Two Isoforms of the Essential Argonaute CSR-1 Throughout Caenorhabditis elegans Development.
Charlesworth, Amanda Gene.

Dissecting the Differential Functions of the Two Isoforms of the Essential Argonaute CSR-1 Throughout Caenorhabditis elegans Development. - 1 online resource (218 pages)

Source: Dissertations Abstracts International, Volume: 85-05, Section: B.

Thesis (Ph.D.)--University of Toronto (Canada), 2023.

Includes bibliographical references

Endogenous small RNA pathways play vital roles in regulating gene expression to ensure germline integrity across animal species. The key effectors of these pathways are Argonaute proteins (AGOs), which utilize small RNA partners to identify target transcripts in a sequence-specific manner. The Caenorhabditis elegans genome encodes nineteen functional AGOs that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. Although AGOs predominantly downregulate their targets, the essential C. elegans AGO CSR-1 has been shown to promote and fine-tune germline gene expression. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR1a. Using a series of transgenes (MosSCI), endogenously GFP::3xFLAG-tagged strains (CRISPR/Cas9), and two different antisera that recognize 1) both isoforms of CSR-1 or 2) CSR-1a only, I have identified key differences between the CSR-1 isoforms. I found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR1a plays tissue-specific functions. Although my data also suggest CSR-1a can compensate for CSR-1b's absence if expressed throughout the germline. Small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. CSR-1a integrates into an sRNA regulatory network with spermatogenic AGOs ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. Meanwhile CSR-1b functions alongside the spermatogenic AGOs, but also targets oogenic genes along with WAGO-4. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. My findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9798380841375Subjects--Topical Terms:

583443
Molecular biology.
Subjects--Index Terms:

ArgonautesIndex Terms--Genre/Form:

554714
Electronic books.

Dissecting the Differential Functions of the Two Isoforms of the Essential Argonaute CSR-1 Throughout Caenorhabditis elegans Development.
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520 $a Endogenous small RNA pathways play vital roles in regulating gene expression to ensure germline integrity across animal species. The key effectors of these pathways are Argonaute proteins (AGOs), which utilize small RNA partners to identify target transcripts in a sequence-specific manner. The Caenorhabditis elegans genome encodes nineteen functional AGOs that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. Although AGOs predominantly downregulate their targets, the essential C. elegans AGO CSR-1 has been shown to promote and fine-tune germline gene expression. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR1a. Using a series of transgenes (MosSCI), endogenously GFP::3xFLAG-tagged strains (CRISPR/Cas9), and two different antisera that recognize 1) both isoforms of CSR-1 or 2) CSR-1a only, I have identified key differences between the CSR-1 isoforms. I found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR1a plays tissue-specific functions. Although my data also suggest CSR-1a can compensate for CSR-1b's absence if expressed throughout the germline. Small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. CSR-1a integrates into an sRNA regulatory network with spermatogenic AGOs ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. Meanwhile CSR-1b functions alongside the spermatogenic AGOs, but also targets oogenic genes along with WAGO-4. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. My findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.
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