CCAR-1 works together with the U2AF large subunit UAF-1 to regulate alternative splicing.

The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members have recently emerged as regulators of alternative splicing and transcription, as well as having other key physiological functions. For example, mammalian CCAR2/DBC1 forms a complex with the zinc factor protein ZNF326 to integrate alternative splicing with RNA polymerase II transcriptional elongation in AT-rich regions of the DNA. Additionally, Caenorhabditis elegans CCAR-1, a homolog to mammalian CCAR2, facilitates the alternative splicing of the perlecan unc-52 gene. However, much about the CCAR family's role in alternative splicing is unknown. Here, we have examined the role of CCAR-1 in genome-wide alternative splicing in Caenorhabditis elegans and have identified new alternative splicing targets of CCAR-1 using RNA sequencing. Also, we found that CCAR-1 interacts with the spliceosome factors UAF-1 and UAF-2 using mass spectrometry, and that knockdown of ccar-1 affects alternative splicing patterns, motility, and proteostasis of UAF-1 mutant worms. Collectively, we demonstrate the role of CCAR-1 in regulating global alternative splicing in C. elegans and in conjunction with UAF-1.

The CBP-1/p300 Lysine Acetyltransferase Regulates the Heat Shock Response in C. elegans.

The decline of proteostasis is a hallmark of aging that is, in part, affected by the dysregulation of the heat shock response (HSR), a highly conserved cellular response to proteotoxic stress in the cell. The heat shock transcription factor HSF-1 is well-studied as a key regulator of proteostasis, but mechanisms that could be used to modulate HSF-1 function to enhance proteostasis during aging are largely unknown. In this study, we examined lysine acetyltransferase regulation of the HSR and HSF-1 in C. elegans. We performed an RNA interference screen of lysine acetyltransferases and examined mRNA expression of the heat-shock inducible gene hsp-16.2, a widely used marker for HSR activation. From this screen, we identified one acetyltransferase, CBP-1, the C. elegans homolog of mammalian CREB-binding protein CBP/p300, as a negative regulator of the HSR. We found that while knockdown of CBP-1 decreases the overall lifespan of the worm, it also enhances heat shock protein production upon heat shock and increases thermotolerance of the worm in an HSF-1 dependent manner. Similarly, we examined a hallmark of HSF-1 activation, the formation of nuclear stress bodies (nSBs). In analyzing the recovery rate of nSBs, we found that knockdown of CBP-1 enhanced the recovery and resolution of nSBs after stress. Collectively, our studies demonstrate a role of CBP-1 as a negative regulator of HSF-1 activity and its physiological effects at the organismal level upon stress.

Cellular Proteomes Drive Tissue-Specific Regulation of the Heat Shock Response.

The heat shock response (HSR) is a cellular stress response that senses protein misfolding and restores protein folding homeostasis, or proteostasis. We previously identified an HSR regulatory network in Caenorhabditis elegans consisting of highly conserved genes that have important cellular roles in maintaining proteostasis. Unexpectedly, the effects of these genes on the HSR are distinctly tissue-specific. Here, we explore this apparent discrepancy and find that muscle-specific regulation of the HSR by the TRiC/CCT chaperonin is not driven by an enrichment of TRiC/CCT in muscle, but rather by the levels of one of its most abundant substrates, actin. Knockdown of actin subunits reduces induction of the HSR in muscle upon TRiC/CCT knockdown; conversely, overexpression of an actin subunit sensitizes the intestine so that it induces the HSR upon TRiC/CCT knockdown. Similarly, intestine-specific HSR regulation by the signal recognition particle (SRP), a component of the secretory pathway, is driven by the vitellogenins, some of the most abundant secretory proteins. Together, these data indicate that the specific protein folding requirements from the unique cellular proteomes sensitizes each tissue to disruption of distinct subsets of the proteostasis network. These findings are relevant for tissue-specific, HSR-associated human diseases such as cancer and neurodegenerative diseases. Additionally, we characterize organismal phenotypes of actin overexpression including a shortened lifespan, supporting a recent hypothesis that maintenance of the actin cytoskeleton is an important factor for longevity.