CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

Stress-induced transcriptional readthrough into neighboring genes is linked to intron retention.

Exposure to certain stresses leads to readthrough transcription. Using polyA-selected RNA-seq in mouse fibroblasts subjected to heat shock, oxidative, or osmotic stress, we found that readthrough transcription can proceed into proximal downstream genes, in a phenomenon previously termed "read-in." We found that read-in genes share distinctive genomic characteristics; they are GC-rich and extremely short , with genomic features conserved in human. Using ribosome profiling, we found that read-in genes show significantly reduced translation. Strikingly, read-in genes demonstrate marked intron retention, mostly in their first introns, which could not be explained solely by their short introns and GC-richness, features often associated with intron retention. Finally, we revealed H3K36me3 enrichment upstream to read-in genes. Moreover, demarcation of exon-intron junctions by H3K36me3 was absent in read-in first introns. Our data portray a relationship between read-in and intron retention, suggesting they may have co-evolved to facilitate reduced translation of read-in genes during stress.

Differential roles for DNAJ isoforms in HTT-polyQ and FUS aggregation modulation revealed by chaperone screens.

Protein aggregation is a hallmark of neurodegeneration. Here, we find that Huntington's disease-related HTT-polyQ aggregation induces a cellular proteotoxic stress response, while ALS-related mutant FUS (mutFUS) aggregation leads to deteriorated proteostasis. Further exploring chaperone function as potential modifiers of pathological aggregation in these contexts, we reveal divergent effects of naturally-occurring chaperone isoforms on different aggregate types. We identify a complex of the full-length (FL) DNAJB14 and DNAJB12, that substantially protects from mutFUS aggregation, in an HSP70-dependent manner. Their naturally-occurring short isoforms, however, do not form a complex, and lose their ability to preclude mutFUS aggregation. In contrast, DNAJB12-short alleviates, while DNAJB12-FL aggravates, HTT-polyQ aggregation. DNAJB14-FL expression increases the mobility of mutFUS aggregates, and restores the deteriorated proteostasis in mutFUS aggregate-containing cells and primary neurons. Our results highlight a maladaptive cellular response to pathological aggregation, and reveal a layer of chaperone network complexity conferred by DNAJ isoforms, in regulation of different aggregate types.