Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels

Nussbacher, Julia K.; Yeo, G

doi:10.1016/j.molcel.2018.02.012

Cited by 116 publications

(117 citation statements)

References 41 publications

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“…The ENCODE RNA binding protein resource contains 1,223 replicated datasets for 356 RBPs, including in vivo targets by eCLIP, in vitro binding motifs by RNA Bind-N-Seq, subcellular localization by immunofluorescence, factor-responsive expression and splicing changes by knockdown/RNA-seq, and DNA associations by ChIP-seq [20]. This unique resource has already proven useful in characterizing allele-specific RBP interactions [51,52], identify candidate regulators of miRNA processing [53], predicting whether RNAs are protein-coding or non-coding [54], and identifying novel factors which act to suppress improper RNA processing caused by retrotransposable elements [36], and will continue to enable researchers to ask broad questions about basic RNA processing mechanisms, deeply consider the functional roles of an individual RBP, or even query an RNA of interest in order to gain insight into potential regulators. Here we describe examples how integrated analyses of binding profiles obtained from eCLIP can yield novel insights into both processing of standard mRNAs as well as other RNA families, including identifying new characteristics of ribosomal RNA processing and the role of RBP interactions with retrotransposable elements.…”

Section: Discussionmentioning

confidence: 99%

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Nostrand

Pratt

Yee

et al. 2019

Preprint

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AbstractA critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enabled mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale integrated analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the large-scale mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Here we describe our analysis of 223 enhanced (eCLIP) datasets characterizing 150 RBPs in K562 and HepG2 cell lines, revealing a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multi-copy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation (enabling identification of branch points with single-nucleotide resolution) and provides genome-wide validation for a branch point-based scanning model for 3’ splice site recognition. Further, we show that eCLIP peak co-occurrences across RBPs enables the discovery of novel co-interacting RBPs. Finally, we present a protocol for visualization of RBP:RNA complexes in the eCLIP workflow using biotin and standard chemiluminescent visualization reagents, enabling simplified confirmation of ribonucleoprotein enrichment without radioactivity. This work illustrates the value of integrated analysis across eCLIP profiling of RBPs with widely distinct functions to reveal novel RNA biology. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

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Section: Discussionmentioning

confidence: 99%

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Nostrand

Pratt

Yee

et al. 2019

Preprint

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“…This is likely facilitated by the recruitment of functionally-related miRNAs into specific miRNA-RBP ternary complexes (e.g. TUG1-hnRNPA1-SET/CCND2/CDK6) 59,61,74 . In this regard, we showed that 15 validated TUG1-interacting miRNAs are differentially expressed in CD34 + CD38and CD34 + CD38 + CML (CP and BC) BM cells and have defined roles in solid tumors and AML/CML stem/progenitor cells 75 (Supplementary Fig.…”

Section: Biologic and Therapeutic Relevance Of The Mir300-tug1 Interpmentioning

confidence: 99%

The 14q32.31DLK1-DIO3 MIR300 tumor suppressorpromotes leukemogenesis by inducing cancer stem cell quiescence and inhibiting NK cell anti-cancer immunity

Silvestri

Trotta

Stramucci

et al. 2019

Preprint

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Drug-resistance of tumor-initiating cells, impaired NK cell immune-response, PP2A loss-offunction and aberrant miRNA expression are cancer features resulting from microenvironmentaland tumor-specific signals. Here we report that genomic-imprinted MIR300 is a cell contextindependent dual function tumor suppressor which is upregulated in quiescent leukemic stem (LSC) and NK cells by microenvironmental signals to induce quiescence and impair immuneresponse, respectively, but inhibited in CML and AML proliferating blasts to prevent PP2Ainduced apoptosis. MIR300 anti-proliferative and PP2A-activating functions are differentially activated through dose-dependent CCND2/CDK6 and SET inhibition, respectively. LSCs escape PP2A-mediated apoptosis through TUG1 lncRNA that uncouples and limits MIR300 functions to cytostasis by regulating unbound-MIR300 levels. Halting MIR300 homeostasis restores NK cell activity and suppresses leukemic but not normal hematopoiesis by eradicating nearly all LSCs.Thus, MIR300 tumor suppressor activity is essential and therapeutically important for LSC-driven leukemias.

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“…Notably, not all human pri-miRNAs contain such motifs, suggesting that other factors may enable Microprocessor-mediated processing. Indeed, various accessory proteins have been reported to either bind directly to the pri-miRNA or to the Microprocessor itself , thereby assisting in pri-miRNA cleavage (Alarcón et al, 2015;Cheng et al, 2014;Fletcher et al, 2016;Nussbacher and Yeo, 2018;Treiber et al, 2017;Tu et al, 2015). Moreover, recent studies also reveal stem-loop structures as cis-regulatory elements in pri-miRNA processing.…”

Section: Introductionmentioning

confidence: 99%

SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts

Hutter

Lohmueller

Jukic

et al. 2019

Preprint

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Highlights• the primary miR-15a stem-loop structure per se is a poor Microprocessor substrate• cleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNA • sequential pri-miRNA processing or "cluster assistance" is mediated by SAFB proteins • SAFB2 associates with the Microprocessor Summary MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally silence most protein-coding genes in mammals. They are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent units. Contrasting this view, we here report an unexpected mode of processing of a bicistronic cluster of the miR-15 family, miR-15a-16-1. We find that the primary miR-15a stem-loop is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage, and describe SAFB2 as a novel accessory protein of DROSHA. Notably, SAFB2-mediated cluster assistance expands to other clustered pri-miRNAs including miR-15b, miR-92a and miR-181b, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal DGCR8/DROSHA substrates in clustered primary miRNA transcripts.

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Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels

Cited by 116 publications

References 41 publications

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

The 14q32.31DLK1-DIO3 MIR300 tumor suppressorpromotes leukemogenesis by inducing cancer stem cell quiescence and inhibiting NK cell anti-cancer immunity

SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts

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