RNA m6A modification orchestrates a LINE-1–host interaction that facilitates retrotransposition and contributes to long gene vulnerability

AbstractThe molecular basis underlying the interaction between retrotransposable elements (RTEs) and the human genome remains poorly understood. Here, we profiled N6-methyladenosine (m6A) deposition on nascent RNAs in human cells by developing a new method MINT-Seq, which revealed that many classes of RTE RNAs, particularly intronic LINE-1s (L1s), are strongly methylated. These m6A-marked intronic L1s (MILs) are evolutionarily young, sense-oriented to hosting genes, and are bound by a dozen RNA binding proteins (RBPs) that are putative novel readers of m6A-modified RNAs, including a nuclear matrix protein SAFB. Notably, m6A positively controls the expression of both autonomous L1s and co-transcribed L1 relics, promoting L1 retrotransposition. We showed that MILs preferentially reside in long genes with critical roles in DNA damage repair and sometimes in L1 suppression per se, where they act as transcriptional “roadblocks” to impede the hosting gene expression, revealing a novel host-weakening strategy by the L1s. In counteraction, the host uses the SAFB reader complex to bind m6A-L1s to reduce their levels, and to safeguard hosting gene transcription. Remarkably, our analysis identified thousands of MILs in multiple human fetal tissues, enlisting them as a novel category of cell-type-specific regulatory elements that often compromise transcription of long genes and confer their vulnerability in neurodevelopmental disorders. We propose that this m6A-orchestrated L1–host interaction plays widespread roles in gene regulation, genome integrity, human development and diseases.

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Massively parallel identification of zipcodes in primary cortical neurons

10.1101/2021.10.21.465275 ◽

2021 ◽

Author(s):

Nicolai von Kuegelgen ◽

Samantha Mendonsa ◽

Sayaka Dantsuji ◽

Maya Ron ◽

Marieluise Kirchner ◽

...

Keyword(s):

Cortical Neurons ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Regulatory Elements ◽

Massively Parallel ◽

Primary Cortical Neurons ◽

Subcellular Compartments ◽

Local Functions ◽

Massively Parallel Reporter Assay

Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

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Functional characterization of splicing regulatory elements

10.1101/2021.05.14.444228 ◽

2021 ◽

Author(s):

Scott I Adamson ◽

Lijun Zhan ◽

Brenton R Graveley

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Functional Characterization ◽

Regulatory Elements ◽

Small Subset ◽

General Sequence ◽

Splicing Regulators ◽

Splicing Regulatory Elements

Background: RNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high-throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms, and in particular, the mediating RNA binding proteins, underlying these associations. Results: Here we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins. We identify features which make splicing events more sensitive to depletion of RNA binding proteins, as well as which RNA binding proteins act as splicing regulators sensitive to depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RNA binding proteins which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied. Conclusions: This study identifies sequence and other features underlying splicing regulation mediated specific RNA binding proteins, as well as validates and identifies other potentially important regulators of splicing in other large transcriptomic datasets.

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Deep analysis of RNA N6-adenosine methylation (m6A) patterns in human cells

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqaa007 ◽

2020 ◽

Vol 2 (1) ◽

Author(s):

Jun Wang ◽

Liangjiang Wang

Keyword(s):

Rna Binding ◽

Data Transfer ◽

Rna Binding Proteins ◽

Consensus Sequence ◽

Model Performance ◽

Cell Types ◽

Human Cells ◽

Rna Modification ◽

Cell Type ◽

Cell Type Specific

Abstract N6-adenosine methylation (m6A) is the most abundant internal RNA modification in eukaryotes, and affects RNA metabolism and non-coding RNA function. Previous studies suggest that m6A modifications in mammals occur on the consensus sequence DRACH (D = A/G/U, R = A/G, H = A/C/U). However, only about 10% of such adenosines can be m6A-methylated, and the underlying sequence determinants are still unclear. Notably, the regulation of m6A modifications can be cell-type-specific. In this study, we have developed a deep learning model, called TDm6A, to predict RNA m6A modifications in human cells. For cell types with limited availability of m6A data, transfer learning may be used to enhance TDm6A model performance. We show that TDm6A can learn common and cell-type-specific motifs, some of which are associated with RNA-binding proteins previously reported to be m6A readers or anti-readers. In addition, we have used TDm6A to predict m6A sites on human long non-coding RNAs (lncRNAs) for selection of candidates with high levels of m6A modifications. The results provide new insights into m6A modifications on human protein-coding and non-coding transcripts.

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The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

eLife ◽

10.7554/elife.09268 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 43

Author(s):

Anthony J Linares ◽

Chia-Ho Lin ◽

Andrey Damianov ◽

Katrina L Adams ◽

Bennett G Novitch ◽

...

Keyword(s):

Transcription Factor ◽

Neuronal Development ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Regulatory Elements ◽

Cytoskeletal Proteins ◽

Control Programs ◽

Neuronal Progenitor ◽

Neuronal Genes

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

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High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

10.1101/2021.10.08.463671 ◽

2021 ◽

Author(s):

Mariela Cortés-López ◽

Laura Schulz ◽

Mihaela Enculescu ◽

Claudia Paret ◽

Bea Spiekermann ◽

...

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Single Point ◽

Point Mutations ◽

Therapy Resistance ◽

Regulatory Elements ◽

Splice Isoforms ◽

Exon 2

During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy.

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Prediction of mRNA subcellular localization using deep recurrent neural networks

Bioinformatics ◽

10.1093/bioinformatics/btz337 ◽

2019 ◽

Vol 35 (14) ◽

pp. i333-i342 ◽

Cited By ~ 12

Author(s):

Zichao Yan ◽

Eric Lécuyer ◽

Mathieu Blanchette

Keyword(s):

Subcellular Localization ◽

Messenger Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Elements ◽

Supplementary Information ◽

Specific Sequence ◽

Structure Information ◽

Subcellular Compartments ◽

Sequence Elements

Abstract Motivation Messenger RNA subcellular localization mechanisms play a crucial role in post-transcriptional gene regulation. This trafficking is mediated by trans-acting RNA-binding proteins interacting with cis-regulatory elements called zipcodes. While new sequencing-based technologies allow the high-throughput identification of RNAs localized to specific subcellular compartments, the precise mechanisms at play, and their dependency on specific sequence elements, remain poorly understood. Results We introduce RNATracker, a novel deep neural network built to predict, from their sequence alone, the distributions of mRNA transcripts over a predefined set of subcellular compartments. RNATracker integrates several state-of-the-art deep learning techniques (e.g. CNN, LSTM and attention layers) and can make use of both sequence and secondary structure information. We report on a variety of evaluations showing RNATracker’s strong predictive power, which is significantly superior to a variety of baseline predictors. Despite its complexity, several aspects of the model can be isolated to yield valuable, testable mechanistic hypotheses, and to locate candidate zipcode sequences within transcripts. Availability and implementation Code and data can be accessed at https://www.github.com/HarveyYan/RNATracker. Supplementary information Supplementary data are available at Bioinformatics online.

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Functional reconstruction of NANOS3 expression in the germ cell lineage by a novel transgenic reporter reveals distinct subcellular localizations of NANOS3

Reproduction ◽

10.1530/rep-09-0373 ◽

2010 ◽

Vol 139 (2) ◽

pp. 381-393 ◽

Cited By ~ 10

Author(s):

Masashi Yamaji ◽

Takashi Tanaka ◽

Mayo Shigeta ◽

Shinichiro Chuma ◽

Yumiko Saga ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Regulatory Elements ◽

Initiation Factor ◽

Processing Bodies

Mutations of RNA-binding proteins such as NANOS3, TIAL1, and DND1 in mice have been known to result in the failure of survival and/or proliferation of primordial germ cells (PGCs) soon after their fate is specified (around embryonic day (E) 8.0), leading to the infertility of these animals. However, the mechanisms of actions of these RNA-binding proteins remain largely unresolved. As a foundation to explore the role of these RNA-binding proteins in germ cells, we established a novel transgenic reporter strain that expresses NANOS3 fused with EGFP under the control of Nanos3 regulatory elements. NANOS3–EGFP exhibited exclusive expression in PGCs as early as E7.25, and continued to be expressed in female germ cells until around E14.5 and in male germ cells throughout the fetal period with declining expression levels after E16.5. NANOS3–EGFP resumed strong expression in postnatal spermatogonia and continued to be expressed in undifferentiated spermatogonial cells in adults. Importantly, the Nanos3–EGFP transgene rescued the sterile phenotype of Nanos3 homozygous mutants, demonstrating the functional equivalency of NANOS3–EGFP with endogenous NANOS3. We found that throughout germ cell development, a predominant amount of NANOS3–EGFP co-localized with TIAL1 (also known as TIAR) and phosphorylated eukaryotic initiation factor 2α, markers for the stress granules, whereas a fraction of it showed co-localization with DCP1A, a marker for the processing bodies. On the other hand, NANOS3–EGFP did not co-localize with Tudor domain-containing protein 1, a marker for the intermitochondrial cements, in spermatogenic cells. These findings unveil the presence of distinct posttranscriptional regulations in PGCs soon after their specification, for which RNA-binding proteins such as NANOS3 and TIAL1 would play critical functions.

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POS-1 and GLD-1 repress glp-1 translation through a conserved binding-site cluster

Molecular Biology of the Cell ◽

10.1091/mbc.e12-03-0216 ◽

2012 ◽

Vol 23 (23) ◽

pp. 4473-4483 ◽

Cited By ~ 15

Author(s):

Brian M. Farley ◽

Sean P. Ryder

Keyword(s):

Cell Fate ◽

Binding Sites ◽

Rna Binding ◽

Zinc Finger Protein ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Regulatory Elements ◽

Regulatory Sequence ◽

Regulatory Pathways ◽

Fate Specification

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.

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Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502849112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3445-3450 ◽

Cited By ~ 84

Author(s):

Qinghong Yan ◽

Sebastien M. Weyn-Vanhentenryck ◽

Jie Wu ◽

Steven A. Sloan ◽

Ye Zhang ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Splice Variants ◽

Mammalian Brain ◽

Cell Type ◽

Evolutionary Selection ◽

Chromatin Regulators ◽

Brain Transcriptome ◽

Cell Type Specific ◽

Specific Regulation

Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23–42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.

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HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

10.1101/175901 ◽

2017 ◽

Author(s):

Jonathan M. Howard ◽

Hai Lin ◽

Garam Kim ◽

Jolene M Draper ◽

Maximilian Haeussler ◽

...

Keyword(s):

Splice Site ◽

Binding Sites ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Elements ◽

Splice Sites ◽

Spliceosome Assembly ◽

Over Expression

AbstractAlternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3’ splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3’ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to “decoy” binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3’ splice sites.

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