Nejc Haberman

Imperial College London | Imperial · Department of Brain Sciences

The 3’ untranslated region (3’UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5’ ends of mRNAs, additionally reveals a large number of apparently 5’ capped RNAs derived from 3’UTRs. Here we provide the first direct evidence...

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic envir...

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyade...

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Aim/Hypothesis Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of beta cell development and function. Here, we investigate roles for an antisense lncRNA expressed from the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in beta cell identity and functionality. Methods Pax6os1 expression was silenced in MIN6 cells...

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal c...

Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. Here we developed a metho...

Many nascent long non-coding RNAs (lncRNAs) undergo the same maturation steps as pre-mRNAs of protein-coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences using the ncRNA-a2 as a model. Genome-wide analyses of intergenic lncRNAs (lin...

Splicing-dependent assembly of the exon junction complex (EJC) at canonical sites -20 to -24 nucleotides upstream of exon-exon junctions in mRNAs occurs in all higher eukaryotes and affects most major regulatory events in the life of a transcript. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila the...

Recursive splicing (RS) starts by defining an “RS-exon,” which is then spliced to the preceding exon, thus creating a recursive 5′ splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the...

Table S7. Accession Numbers for RNA-Seq Data from the ENCODE Consortium Used in Figure 2B

Table S5. Sequences of the Splicing Reporters Used in this Study, Related to Figures 1, 3, 4, and 5

Table S4. Summary Statistics of mRNA 3-End Sequencing Experiments, Related to Figure 3 This table summarizes the analysis of changes in polyA site usage (pairs of pA1 and pA2), and if any of the two polyA sites originates from inside a LINE repeat.

Table S6. Inclusion Levels of 43583 UCSC Annotated Exons in 53 Human Tissue Types, Related to Figure 5 Repeat-derived exons were defined as any exon, for which either the 5′ or the 3′ splice site is derived from an Alu or a LINE repeat. We quantified these and all exons of the same genes in the V6p data of the GTEx consortium. In total, we covered...

Table S7. Summary Statistics of Pentamer Frequencies in Antisense L1 Sequences, Related to Figure 6 This table provides summary statistics on the frequency of every pentamer within antisense L1 repeats. Column 1 contains the pentamer itself, column 2 the percentage of repeats that have at least one occurrence of the sequence, column 3 the median c...

Table S1. Sources and References for CLIP and RNA-Seq Datasets, as Well as RBP Binding Motifs, Related to STAR Methods This file contains 6 spreadsheets (tabs) in total, all dedicated to provide the source of data used to generate the main figures; including accession number of published iCLIP, eCLIP and RNaseq data. Tab 4 contains the motifs we c...

Table S2. Quantification of L1 and L2 Sequences in iCLIP and eCLIP, Related to Figure 1 This table contains the percentage of reads we found to map to L1 and L2 sequences in sense or in antisense in each data set.

Table S3. Summary Statistics of Cryptic Exon Annotation Based on UCSC Annotation and Cufflinks Assembly, Related to Figure 3 This table explains our definition of cryptic exons, and the number of LINE-derived exons at 15% inclusion level.

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs r...

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein–RNA interactions. UV crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that copurify with a selected RNA-binding protein under stringent conditions. Here we focus on approach...

Epithelial-resident T lymphocytes, such as intraepithelial lymphocytes (IELs) located at the intestinal barrier, can offer swift protection against invading pathogens. Lymphocyte activation is strictly regulated because of its potential harmful nature and metabolic cost, and most lymphocytes are maintained in a quiescent state. However, IELs are ke...

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to simultaneously map human spliceosome engagement with snRNAs and pre-mRNAs. We identify nine sites on pre-mRNAs that overlap with position-d...

We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of t...

It is challenging for RNA processing machineries to select exons within long intronic regions. We find that intronic LINE repeat sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs). This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites in LINEs. Both RBPs repress splicing an...

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein–RNA interactions. UV crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that copurify with a selected RNA-binding protein under stringent conditions. Here we focus on approach...

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein-RNA interactions. Crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that co-purify with a selected RBP under stringent conditions. Here we focus on approaches for the analysi...

RNA-binding proteins (RBPs) are the primary regulators of all aspects of posttranscriptional gene regulation. In order to understand how RBPs perform their function, it is important to identify their binding sites. Recently, new techniques have been developed to employ high-throughput sequencing to study protein-RNA interactions in vivo, including...

Upon publication of the original article, it was noted that data submitted by the authors was accidentally omitted during typesetting. The following section of the submitted manuscript was wrongfully omitted from the Methods section in the published article.

Table S3. Complete Table of All Analyzed Genes with Information about the Selected Proximal and Distal Sites for Each Gene, Related to Figures 2 and 3

Table S4. Complete polyA Atlas, All Detected polyA Sites from the 12 Experiments in Our Dataset, with polyAR Classification—Strong, Weak, PAS-less— for Each Site, Related to Figure S2

Detailed information with additional % mapped (unique alignments only) and % of internally primed (reads that were filtered out due to A-richness in the surrounding -5..5 nt region on the reference genome) of Rot et al. and Gruber et al. datasets, Related to Figure S2

Table S1. Detailed Information about the 12 Experimental Datasets Used in Our Study, Related to Figure 2

Category specifies the type and loci of the regulation (enhanced, repressed and proximal, distal).

Table S6. Most Bound and Enriched Fold-Change Regions—50, 75 and 100 nt Windows— Determined from RNA Maps, Related to Figure 7

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3′ mRNA sequencing. This reveals at nucleotide resolution the “RNA maps” des...

Background: Ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) identifies the sites on RNAs that are in direct contact with RNA-binding proteins (RBPs). Several variants of CLIP exist, which require different computational approaches for analysis. This variety of approaches can create challenges for a novice user and can hamper insights...

Percent exon inclusion of Alu-exons in different human tissues.We list percent exon inclusion for each Alu-exon with sufficient coverage across the GTEx data (889 exons with at least 4000 junction-spanning reads across 8555 individual samples). In addition, the data table includes chromosome position of the exon, mean skipping counts, the maximum a...

List of Alu-exons across our datasets and UCSC annotation, including cross-species annotation.We merged all Alu-exons identified from RNAseq data and annotated in UCSC (6731 exons), and filtered for non-overlapping exons. If any two exons overlapped, we retained the larger exon. This created a list of 6309 Alu-exons for which we selected the 3' spl...

List of RT-PCR and RT-qPCR primers used in this study. DOI: http://dx.doi.org/10.7554/eLife.19545.022

Alu elements are retrotransposons that frequently form new exons during primate evolution. Here, we assess the interplay of splicing repression by hnRNPC and nonsense-mediated mRNA decay (NMD) in the quality control and evolution of new Alu-exons. We identify 3100 new Alu-exons and show that NMD more efficiently recognises transcripts with Alu-exon...

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic fai...

It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing...

Nonsense-mediated mRNA decay (NMD) degrades different classes of mRNAs, including transcripts with premature termination codons (PTCs). The NMD factor SMG6 initiates degradation of substrate mRNAs by endonucleolytic cleavage. Here, we aim to delineate the cascade of NMD-activating events that culminate in endocleavage. We report that long 3' UTRs e...

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain,...

Supplementary figures and tables