The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a transcriptional coactivator that controls expression of metabolic/energetic genes, programming cellular responses to nutrient and environmental adaptations such as fasting, cold, or exercise. Unlike other coactivators, PGC-1α contains protein domains involved in RNA regulation such as serine/arginine (SR) and RNA recognition motifs (RRMs). However, the RNA targets of PGC-1α and how they pertain to metabolism are unknown. To address this, we performed enhanced ultraviolet (UV) cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq) in primary hepatocytes induced with glucagon. A large fraction of RNAs bound to PGC-1α were intronic sequences of genes involved in transcriptional, signaling, or metabolic function linked to glucagon and fasting responses, but were not the canonical direct transcriptional PGC-1α targets such as OXPHOS or gluconeogenic genes. Among the top-scoring RNA sequences bound to PGC-1α were Foxo1, Camk1δ, Per1, Klf15, Pln4, Cluh, Trpc5, Gfra1, and Slc25a25 PGC-1α depletion decreased a fraction of these glucagon-induced messenger RNA (mRNA) transcript levels. Importantly, knockdown of several of these genes affected glucagon-dependent glucose production, a PGC-1α-regulated metabolic pathway. These studies show that PGC-1α binds to intronic RNA sequences, some of them controlling transcript levels associated with glucagon action.

Persistent cytoplasmic aggregates containing RNA binding proteins (RBPs) are central to the pathogenesis of late-onset neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). These aggregates share components, molecular mechanisms, and cellular protein quality control pathways with stress-induced RNA granules (SGs). Here, we assess the impact of stress on the global mRNA localization landscape of human pluripotent stem cell-derived motor neurons (PSC-MNs) using subcellular fractionation with RNA sequencing and proteomics. Transient stress disrupts subcellular RNA and protein distributions, alters the RNA binding profile of SG- and ALS-relevant RBPs and recapitulates disease-associated molecular changes such as aberrant splicing of STMN2. Although neurotypical PSC-MNs re-establish a normal subcellular localization landscape upon recovery from stress, cells harboring ALS-linked mutations are intransigent and display a delayed-onset increase in neuronal cell death. Our results highlight subcellular molecular distributions as predictive features and underscore the utility of cellular stress as a paradigm to study ALS-relevant mechanisms.

Background

Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts.

Methods

We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq.

Results

We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients.

Conclusions

Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles.

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

The N6-methyladenosine (m⁶A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m⁶A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Background

Infection prevention (IP) measures are designed to mitigate the transmission of pathogens in healthcare. Using large-scale viral genomic and social network analyses, we determined if IP measures used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic were adequate in protecting healthcare workers (HCWs) and patients from acquiring SARS-CoV-2.

Methods

We performed retrospective cross-sectional analyses of viral genomics from all available SARS-CoV-2 viral samples collected at UC San Diego Health and social network analysis using the electronic medical record to derive temporospatial overlap of infections among related viromes and supplemented with contact tracing data. The outcome measure was any instance of healthcare transmission, defined as cases with closely related viral genomes and epidemiological connection within the healthcare setting during the infection window. Between November 2020 through January 2022, 12 933 viral genomes were obtained from 35 666 patients and HCWs.

Results

Among 5112 SARS-CoV-2 viral samples sequenced from the second and third waves of SARS-CoV-2 (pre-Omicron), 291 pairs were derived from persons with a plausible healthcare overlap. Of these, 34 pairs (12%) were phylogenetically linked: 19 attributable to household and 14 to healthcare transmission. During the Omicron wave, 2106 contact pairs among 7821 sequences resulted in 120 (6%) related pairs among 32 clusters, of which 10 were consistent with healthcare transmission. Transmission was more likely to occur in shared spaces in the older hospital compared with the newer hospital (2.54 vs 0.63 transmission events per 1000 admissions, P < .001).

Conclusions

IP strategies were effective at identifying and preventing healthcare SARS-CoV-2 transmission.

A promising approach to help students safely return to in person learning is through the application of sentinel cards for accurate high resolution environmental monitoring of SARS-CoV-2 traces indoors. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface materials, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, Wet Wipes, RNase Away) at three different viral loads: "high" (4 × 10⁴ GE/mL), "medium" (1 × 10⁴ GE/mL), and "low" (2.5 × 10³ GE/mL). RNase Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet Wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. IMPORTANCE Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are a persistent signal or new infectious cases and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.