Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

Abstract

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.

Fig. 1.

RNAi for hsp-1 (F26D10.3) and hsf-1 (Y53C10.A12) induces aggregate formation of polyglutamine YFP proteins in a Q stretch length-dependent manner. (A–O) Fluorescence microscopy pictures of 4-day-old C. elegans expressing polyglutamine YFP with Q stretches of 0 (A, F, and K), 24 (B, G, and L), 33 (C, H, and M), 35 (D, I, and N), and 40 residues (E, J, and O). Animals were synchronized to the L1 stage and fed for 72 h with bacteria containing an empty vector (A–E), double-stranded RNA (dsRNA) against hsp-1 (F–J), or dsRNA against hsf-1 (K–O). (Bar = 50 μm.). (P–U) Aggregates of Q35 YFP induced by RNAi for hsp-1 or hsf-1 are biophysically similar to aggregates of Q40-YFP. FRAP analysis of the mobility of diffusely stained (P and R) or aggregated (Q, S, and T) Q0 YFP (P), Q40 YFP (Q), or Q35 YFP (R–T) fed with control bacteria (R), bacteria expressing dsRNA against hsp-1 (S), or bacteria expressing dsRNA against hsf-1 (T). (Bar = 2 μm.) Fluorescent molecules in the boxed area (white box) were subjected to FRAP analysis. Single scanned images were taken before (prebleach) and at the indicated time points after photobleaching. (U) Quantitative FRAP analysis of soluble or aggregate-associated YFP fusion proteins. The relative fluorescence intensity was determined for each time point and was represented as the average of the analysis of three independent measurements. Error bars indicate SEM. (V) Aggregates of Q35 YFP induced by RNAi for hsp-1 or hsf-1 are biochemically similar to aggregates of Q40 YFP. Extracts of animals expressing YFP fusion proteins were separated by SDS/PAGE and immunoblotted with an anti-YFP antibody. Lysates of Q0 animals (lane 1), lysates of Q35 animals or Q24 animals fed with empty vector (lanes 2 and 5), hsp-1 RNAi food (lane 3 and 6), or hsf-1 RNAi food (lanes 4 and 7). Asterisk indicates degradation products.

Fig. 2.

RNAi for newly identified modifiers of polyglutamine aggregate formation induces aggregates and insolubilization of polyglutamine proteins that depend on the length of the Q stretch. (A–C) Fluorescence microscopy pictures of 4-day-old C. elegans expressing Q35 YFP fed with control RNAi food (A) and two examples of positive clones expressing dsRNA against hsp-6 (B) and snr-4 (C). (Bar = 50 μm.) (D) Western blot analysis of extracts of animals expressing Q24 YFP or Q35 YFP, fed with bacteria expressing dsRNA for the indicated genes. Genes are grouped according to their functional classes.

Fig. 3.

Overview of modifiers of polyglutamine aggregation. Represented are the five major classes of genes of which knock-down by RNAi enhances polyglutamine aggregate formation. We propose that knock-down of genes in these classes induces aggregate formation either by an increase in the production of misfolded proteins (green line) or by a decrease in the clearance of misfolded proteins (red line). Between brackets is the number of genes found in the indicated functional class.