Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Summary

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Abstract

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters and behavioral assays function as valuable readouts that enable the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Introduction

Cellular proteostasis is monitored by an intricate network of protein quality control components such as molecular chaperones, stress responses and degradation mechanisms including the ubiquitin proteasome system (UPS) and autophagy^1,2. The activation of stress response pathways, such as the HSF-1 mediated heat shock response (HSR), the unfolded protein response of the endoplasmic reticulum (UPR^ER) and the mitochondria (UPR^mito) is vital for cellular adaptation to and survival during environmental challenges or protein misfolding disease that lead to toxic protein aggregation

Protocol

1. Tissue-specific RNAi in two ways: Hairpin RNAi and tissue-specific SID-1 complementation

1. Generation of hairpin RNAi constructs for tissue-specific expression in sid-1 mutants
   1. Amplify the target gene sequence (e.g., hsp-90 sequence isolated from the hsp-90 RNAi clone from the Ahringer RNAi library²⁰) by PCR. Place a nonpalindromic sequence at the 3’ end of the hsp-90 sequence, that is a SfiI site (ATCTA)²¹........

Discussion

The methods described here demonstrate the use of tools that allow for the tissue-specific knockdown of PN components in a constitutive and temporal manner. We have previously identified TCS, a cell nonautonomous stress response mechanism that is induced by tissue-specific alteration of Hsp90 expression levels¹⁰. Tissue-specific knockdown of hsp-90 by expression of hairpin RNAi leads to cell nonautonomous upregulation of protective hsp-70 chaperone expression in distal tissues, t.......

Materials

Name	Company	Catalog Number	Comments
Ampicillin	Merck	A0166-5G	Protocol Section 3.1.
DNA Clean & Concentrator-500	Zymo Research	D4031	Protocol Section 2.2.
IPTG Isopropyl-β-D-thiogalactoside	Merck	367-93-1	Protocol Section 3.1.
Multisite Gateway Cloning Kit	Thermo Fisher	12537100	Protocol Section 1.2.
SigmaCote	Merck	SL2-25mL	Protocol Section 2.3.
Tetracycline	Merck	T7660-5G	Protocol Section 3.1.
Zero Blunt TOPO PCR Cloning Kit	Thermo Fisher	K280002	Protocol Section 1.1.