Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer.

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma.

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.

Dissecting the biophysics and biology of intrinsically disordered proteins.

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.

ZNF692 organizes a hub specialized in 40S ribosomal subunit maturation enhancing translation in rapidly proliferating cells.

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Protein arginine methyltransferase 5 (PRMT5) is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer.

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer acquired resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Using a genome-wide CRISPR screen, we identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout (RBKO) breast cancer cells. PRMT5 inhibition blocked cell cycle G1-to-S transition independent of RB, thus arresting growth of RBKO cells. Proteomics analysis uncovered fused in sarcoma (FUS) as a downstream effector of PRMT5. Pharmacological inhibition of PRMT5 resulted in dissociation of FUS from RNA polymerase II (Pol II), Ser2 Pol II hyperphosphorylation, and intron retention in genes that promote DNA synthesis. Treatment with the PRMT5i inhibitor pemrametostat and fulvestrant synergistically inhibited growth of ER+/RB-deficient patient-derived xenografts, suggesting dual ER and PRMT5 blockade as a novel therapeutic strategy to treat ER+/RB-deficient breast cancer.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8 , which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer.

Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated in the mutational signatures of more than 70% of human cancers. However, the mechanism underlying how cancer cells hijack the APOBEC mediated mutagenesis machinery to promote tumor heterogeneity, and thereby foster therapy resistance remains unclear. We identify SYNCRIP as an endogenous molecular brake which suppresses APOBEC-driven mutagenesis in prostate cancer (PCa). Overactivated APOBEC3B, in SYNCRIP-deficient PCa cells, is a key mutator, representing the molecular source of driver mutations in some frequently mutated genes in PCa, including FOXA1, EP300. Functional screening identifies eight crucial drivers for androgen receptor (AR)-targeted therapy resistance in PCa that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. These results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring AR-targeted therapy resistance in PCa.

Localization of Kif1c mRNA to cell protrusions dictates binding partner specificity of the encoded protein.

Subcellular localization of messenger RNA (mRNA) is a widespread phenomenon that can impact the regulation and function of the encoded protein. In nonneuronal cells, specific mRNAs localize to cell protrusions, and proper mRNA localization is required for cell migration. However, the mechanisms by which mRNA localization regulates protein function in this setting remain unclear. Here, we examined the functional consequences of localization of the mRNA encoding KIF1C. KIF1C is a kinesin motor protein required for cell migration and mRNA trafficking, including trafficking of its own mRNA. We show that Kif1c mRNA localization does not regulate KIF1C's protein abundance, distribution, or ability to traffic other mRNAs. Conversely, Kif1c mRNA localization to protrusions is required for directed cell migration. We used mass spectrometry to identify binding partners of endogenous KIF1C, which revealed dramatic dysregulation of the number and identity of KIF1C interactors in response to Kif1c mRNA mislocalization. These results therefore uncovered a mechanistic connection between mRNA localization to cell protrusions and the specificity of protein-protein interactions. We anticipate that this mechanism is not limited to Kif1c and is likely to be a general principle that impacts the functions of proteins encoded by protrusion-enriched mRNAs in nonneuronal cells.

CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.

During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.

Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

MicroRNA turnover: a tale of tailing, trimming, and targets.

MicroRNAs (miRNAs) post-transcriptionally repress gene expression by guiding Argonaute (AGO) proteins to target mRNAs. While much is known about the regulation of miRNA biogenesis, miRNA degradation pathways are comparatively poorly understood. Although miRNAs generally exhibit slow turnover, they can be rapidly degraded through regulated mechanisms that act in a context- or sequence-specific manner. Recent work has revealed a particularly important role for specialized target interactions in controlling rates of miRNA degradation. Engagement of these targets is associated with the addition and removal of nucleotides from the 3' ends of miRNAs, a process known as tailing and trimming. Here we review these mechanisms of miRNA modification and turnover, highlighting the contexts in which they impact miRNA stability and discussing important questions that remain unanswered.

RBM33 directs the nuclear export of transcripts containing GC-rich elements.

Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX-NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

NORAD-induced Pumilio phase separation is required for genome stability.

Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization1,2. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism3,4, whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins5-8. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs9-11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.

A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming.

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. We identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

Ribosome Recycling by ABCE1 Links Lysosomal Function and Iron Homeostasis to 3' UTR-Directed Regulation and Nonsense-Mediated Decay.

Nonsense-mediated decay (NMD) is a pathway that degrades mRNAs containing premature termination codons. Here we describe a genome-wide screen for NMD factors that uncovers an unexpected mechanism that broadly governs 3' untranslated region (UTR)-directed regulation. The screen reveals that NMD requires lysosomal acidification, which allows transferrin-mediated iron uptake, which, in turn, is necessary for iron-sulfur (Fe-S) cluster biogenesis. This pathway maintains the activity of the Fe-S cluster-containing ribosome recycling factor ABCE1, whose impaired function results in movement of ribosomes into 3' UTRs, where they displace exon junction complexes, abrogating NMD. Importantly, these effects extend beyond NMD substrates, with ABCE1 activity required to maintain the accessibility of 3' UTRs to diverse regulators, including microRNAs and RNA binding proteins. Because of the sensitivity of the Fe-S cluster of ABCE1 to iron availability and reactive oxygen species, these findings reveal an unanticipated vulnerability of 3' UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress.