Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation.

Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIbeta-binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.

Parallel genetics of regulatory sequences using scalable genome editing in vivo.

How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3' UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.

A single dose of the Biontech/Pfizer BNT162b2 vaccine protected elderly residents from severe COVID-19 during a SARS-coronavirus-2 outbreak in a senior citizen home in Germany.

BACKGROUND: A total of 62/66 (93.9%) residents in a senior citizen home in Bremen, Germany, received the first dose of the Biontech/Pfizer vaccine BNT162b2 on December 27th 2020. After routine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen tests showed positive results on January 5th, all residents and staff were tested by RT-PCR. RESULTS: Nine staff members and 23 residents had a positive result. PCR positive staff members reported mild to severe COVID-19 symptoms, one was hospitalized. None of them had been vaccinated. In contrast, the vaccinated residents reported no or only mild symptoms. Sequencing of the SARS-CoV-2 genomes of infected individuals revealed a monophyletic origin of the outbreak within the PANGO lineage B.1.177.86. CONCLUSIONS: In summary, our data show that partial vaccination prevented severe COVID-19 among the residents during this local SARS-CoV-2 outbreak, suggesting a high effectiveness of even a single vaccine dose, but also emphasize that asymptomatic individuals might still be carriers/spreaders.

Identification of proteins and miRNAs that specifically bind an mRNA in vivo.

Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

Post-transcriptional Regulation by 3' UTRs Can Be Masked by Regulatory Elements in 5' UTRs.

In mRNA sequences, 3' UTRs are thought to contain most elements that specifically regulate localization, turnover, and translation. Although high-throughput experiments indicate that many RNA-binding proteins (RBPs) also bind 5' UTRs, much less is known about specific post-transcriptional control exerted by 5' UTRs. GLD-1 is a conserved RBP and a translational repressor with essential roles in Caenorhabditis elegans germ cell development. Previously, we showed that GLD-1 binds highly conserved sites in both 3' and 5' UTRs. Here, by targeted single-copy insertion of transgenes, we systematically tested in vivo functionality of 5' and 3' UTR binding sites individually and in combination. Our data show that sites in 5' UTRs mediate specific and strong translational repression, independent of exact position. Intriguingly, we found that the functionality of 3' UTR sites can be masked by 5' UTR sites and vice versa. We conclude that it is important to study both UTRs simultaneously.

Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection.

BACKGROUND: Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse. RESULTS: Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis. CONCLUSIONS: We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses.