A large-scale binding and functional map of human RNA-binding proteins
Eric L. Van Nostrand, Peter Freese, Gabriel A. Pratt, Xiaofeng Wang, Xintao Wei, Rui Xiao, Steven M. Blue, Jia-Yu Chen, Neal A. L. Cody, Daniel Dominguez, Sara Olson, Balaji Sundararaman, Lijun Zhan, Cassandra Bazile, Louis Philip Benoit Bouvrette, Julie Bergalet, Michael O. Duff, Keri E. Garcia, Chelsea Gelboin-Burkhart, Myles Hochman, Nicole J. Lambert, Hairi Li, Michael P. McGurk, Thai B. Nguyen, Tsultrim Palden, Ines Rabano, Shashank Sathe, Rebecca Stanton, Amanda Su, Ruth Wang, Brian A. Yee, Bing Zhou, Ashley L. Louie, Stefan Aigner , Xiang-Dong Fu, Eric Lécuyer, Christopher B. Burge, Brenton R. Graveley & Gene W. Yeo.
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome . Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).
Van Nostrand EL, Pratt GA, Shishkin, Gelboin-Burkhart C, Fang MY, Sundararaman B, Blue SM, Nguyen TB, Surka C, Elkins K, Stanton R, Rigo F, Guttman M, Yeo GW
As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.
CG14906 (mettl4) mediates m6A methylation of U2 snRNA in Drosophila
While the eukaryotic candidate m6A methyltransferases belong to multiple distinct methylase lineages, the most widespread group belongs to the MT-A70 family exemplified by the yeast messenger RNA (mRNA) adenine methylase complex Ime4/Kar4. At the structural level, all of these enzymes are characterized by a 7-β-strand methyltransferase domain at their C terminus, fused to a predicted α-helical domain at their N terminus and require S-adenosyl-L-methionine (SAM) as a methyl donor. The catalytic motif, [DSH]PP[YFW], present in many members of this family, has shown to be critical for METTL3/METTL4-mediated mRNA m6A methylation1. The high degree of amino acid sequence conservation among the predicted N6-methyladenosine methyltransferases motivates further explorations into their potential functional conservation. METTL4 is a member of the MT-A70-like protein family, which is conserved during evolution (Fig. 1a)2. Previous studies suggested that METTL4 regulates DNA 6mA in vivo and therefore is a candidate DNA 6mA methyltransferase3,4,5. However, the enzymatic activity of METTL4 in vitro has not been demonstrated.
Targeting an RNA-Binding Protein Network in Acute Myeloid Leukemia
Eric Wang, Sydney X. Lu, Alessandro Pastore, Xufeng Chen, Jochen Imig, Stanley Chun-Wei Lee, Kathryn Hockemeyer, Yohana E. Ghebrechristos, Akihide Yoshimi, Daichi Inoue, Michelle Ki, Hana Cho, Lillian Bitner, Andreas Kloetgen, Kuan-Ting Lin, Taisuke Uehara, Takashi Owa, Raoul Tibes, Adrian R. Krainer, Omar Abdel-Wahab, Iannis Aifantis
RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.
The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification
Mona Abed, Erik Verschueren, Hanna Budayeva, Peter Liu, Donald S. Kirkpatrick, Rohit Reja, Sarah K. Kummerfeld, Joshua D. Webster, Sarah Gierke, Mike Reichelt, Keith R. Anderson, Robert J. Newman, Merone Roose-Girma, Zora Modrusan, Hazal Pektas, Emin Maltepe, Kim Newton, Vishva M. Dixit
Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotran- sposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiat- ing TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.