Accurate and precise annotation of 3′ UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding ...protein-mediated mRNA 3′ end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3′ ends and compares favorably with existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo.
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•PAPERCLIP is a high-performance mRNA 3′ end mapping method based on the CLIP technique•PAPERCLIP provides insights into the regulation of alternative polyadenylation•PAPERCLIP expands knowledge of 3′-UTR-mediated mRNA regulation
Hwang et al. develop PAPERCLIP, a high-performance mRNA 3′ end mapping method based on the CLIP technique. They use PAPERCLIP to show that CstF64/64tau promotes non-canonical poly(A) site usage through a GUKKU motif. Furthermore, they also discover shifts in alternative polyadenylation and identify novel microRNA binding sites during mouse brain development.
The extracellular activity of Plasminogen activator inhibitor-1 (PAI-1) is well described, acting as an inhibitor of tissue plasminogen activator and urokinase-type plasminogen activator, impacting ...fibrinolysis. Recent studies have revealed a pro-tumorigenic role of PAI-1 in human cancers, via the regulation of angiogenesis and tumor cell survival. In this study, immunohistochemical staining of 939 human bladder cancer specimens showed that PAI-1 expression levels correlated with tumor grade, tumor stage and overall survival. The typical subcellular localization of PAI-1 is cytoplasmic, but in approximately a quarter of the cases, PAI-1 was observed to be localized to both the tumor cell cytoplasm and the nucleus. To investigate the potential function of nuclear PAI-1 in tumor biology we applied chromatin immunoprecipitation (ChIP)-sequencing, gene expression profiling, and rapid immunoprecipitation mass spectrometry to a pair of bladder cancer cell lines. ChIP-sequencing revealed that PAI-1 can bind DNA at distal intergenic regions, suggesting a role as a transcriptional coregulator. The downregulation of PAI-1 in bladder cancer cell lines caused the upregulation of numerous genes, and the integration of ChIP-sequence and RNA-sequence data identified 57 candidate genes subject to PAI-1 regulation. Taken together, the data suggest that nuclear PAI-1 can influence gene expression programs and support malignancy.
Advances in cancer biology are increasingly dependent on integration of heterogeneous datasets. Large-scale efforts have systematically mapped many aspects of cancer cell biology; however, it remains ...challenging for individual scientists to effectively integrate and understand this data.
We have developed a new data retrieval and indexing framework that allows us to integrate publicly available data from different sources and to combine publicly available data with new or bespoke datasets. Our approach, which we have named the cancer data integrator (CanDI), is straightforward to implement, is well documented, and is continuously updated which should enable individual users to take full advantage of efforts to map cancer cell biology. We show that CanDI empowered testable hypotheses of new synthetic lethal gene pairs, genes associated with sex disparity, and immunotherapy targets in cancer.
CanDI provides a flexible approach for large-scale data integration in cancer research enabling rapid generation of hypotheses. The CanDI data integrator is available at https://github.com/GilbertLabUCSF/CanDI .
Regulatory networks allow organisms to match adaptive behavior to the complex and dynamic contingencies of their native habitats. Upon a sudden transition to a novel environment, the mismatch between ...the native behavior and the new niche provides selective pressure for adaptive evolution through mutations in elements that control gene expression. In the case of core components of cellular regulation and metabolism, with broad control over diverse biological processes, such mutations may have substantial pleiotropic consequences. Through extensive phenotypic analyses, we have characterized the systems-level consequences of one such mutation (rho*) in the global transcriptional terminator Rho of Escherichia coli. We find that a single amino acid change in Rho results in a massive change in the fitness landscape of the cell, with widely discrepant fitness consequences of identical single locus perturbations in rho* versus rho(WT) backgrounds. Our observations reveal the extent to which a single regulatory mutation can transform the entire fitness landscape of the cell, causing a massive change in the interpretation of individual mutations and altering the evolutionary trajectories which may be accessible to a bacterial population.
Drug resistance is a major obstacle in cancer treatment and can involve a variety of different factors. Identifying effective therapies for drug resistant tumors is integral for improving patient ...outcomes.
In this study, we applied a computational drug repositioning approach to identify potential agents to sensitize primary drug resistant breast cancers. We extracted drug resistance profiles from the I-SPY 2 TRIAL, a neoadjuvant trial for early stage breast cancer, by comparing gene expression profiles of responder and non-responder patients stratified into treatments within HR/HER2 receptor subtypes, yielding 17 treatment-subtype pairs. We then used a rank-based pattern-matching strategy to identify compounds in the Connectivity Map, a database of cell line derived drug perturbation profiles, that can reverse these signatures in a breast cancer cell line. We hypothesize that reversing these drug resistance signatures will sensitize tumors to treatment and prolong survival.
We found that few individual genes are shared among the drug resistance profiles of different agents. At the pathway level, however, we found enrichment of immune pathways in the responders in 8 treatments within the HR+HER2+, HR+HER2-, and HR-HER2- receptor subtypes. We also found enrichment of estrogen response pathways in the non-responders in 10 treatments primarily within the hormone receptor positive subtypes. Although most of our drug predictions are unique to treatment arms and receptor subtypes, our drug repositioning pipeline identified the estrogen receptor antagonist fulvestrant as a compound that can potentially reverse resistance across 13/17 of the treatments and receptor subtypes including HR+ and triple negative. While fulvestrant showed limited efficacy when tested in a panel of 5 paclitaxel resistant breast cancer cell lines, it did increase drug response in combination with paclitaxel in HCC-1937, a triple negative breast cancer cell line.
We applied a computational drug repurposing approach to identify potential agents to sensitize drug resistant breast cancers in the I-SPY 2 TRIAL. We identified fulvestrant as a potential drug hit and showed that it increased response in a paclitaxel-resistant triple negative breast cancer cell line, HCC-1937, when treated in combination with paclitaxel.
Circulating tumor cells (CTCs), a population of cancer cells that represent the seeds of metastatic nodules, are a promising model system for studying metastasis. However, the expansion of ...patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here we report the development of a combined CTC and cultured CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. The propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-κB, EMT, and TGFβ signaling as pan-cancer signaling pathways involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective five-gene signature (
,
,
,
, and
). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 (
) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.
Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as ...two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2′-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2′-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite d-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.Two different methylation states of the adenosine adjacent to the snRNA cap are found in the biogenesis process of snRNAs, Am and m6Am, whose levels are regulated by FTO and are related to alternative pre-mRNA splicing.
Microbial biofilms are a dominant feature of many human infections. However, developing effective strategies for controlling biofilms requires an understanding of the underlying biology well beyond ...what currently exists. Using a novel strategy, we have induced formation of a robust biofilm in Escherichia coli by utilizing an exogenous source of poly-N-acetylglucosamine (PNAG) polymer, a major virulence factor of many pathogens. Through microarray profiling of competitive selections, carried out in both transposon insertion and over-expression libraries, we have revealed the genetic basis of PNAG-based biofilm formation. Our observations reveal the dominance of electrostatic interactions between PNAG and surface structures such as lipopolysaccharides. We show that regulatory modulation of these surface structures has significant impact on biofilm formation behavior of the cell. Furthermore, the majority of clinical isolates which produced PNAG also showed the capacity to respond to the exogenously produced version of the polymer.
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