SCIENCE ADVANCES | RESEARCH ARTICLE BIOCHEMISTRY Copyright © 2020 The Authors, some rights reserved; Integrator restrains paraspeckles assembly by exclusive licensee American Association promoting isoform switching of the lncRNA NEAT1 for the Advancement Jasmine Barra1,2,3,4,5, Gabriel S. Gaidosh6, Ezra Blumenthal6, Felipe Beckedorff 6, Mina M. Tayari6, of Science. No claim to 6 7,8 9 3,4,10 3,4 original U.S. Government Nina Kirstein , Tobias K. Karakach , Torben Heick Jensen , Francis Impens , Kris Gevaert , Works. Distributed 5 6 1,2 † Eleonora Leucci *, Ramin Shiekhattar *, Jean-Christophe Marine * under a Creative Commons Attribution RNA 3′ end processing provides a source of transcriptome diversification which affects various (patho)-physiological NonCommercial processes. A prime example is the transcript isoform switch that leads to the read-through expression of the long License 4.0 (CC BY-NC). non-coding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. NEAT1_2 is required for assembly of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that modulate this event, we identified factors involved in the 3′ end processing of polyadenylated RNA and components of the Integrator complex. Perturbation experiments estab- lished that, by promoting the cleavage of NEAT1_2, Integrator forces NEAT1_2 to NEAT1_1 isoform switching and, thereby, restrains PS assembly. Consistently, low levels of Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study establishes that Integrator regulates PS biogenesis and a link between Integrator, cancer biology, and chemosensitivity, which may be exploited therapeutically. INTRODUCTION and/or activity of core 3′ end processing factors can obviously con- Most human genes have multiple sites at which RNA 3′ end cleavage tribute to 3′ end processing rewiring, either globally or specifically, and polyadenylation can occur (1). Alternative 3′ end cleavage gives and thereby affect transcriptome diversification in response to specific rise to transcript isoforms that differ either in their coding sequences environmental cues. Moreover, many other RNA binding proteins or in their 3′ untranslated regions (UTRs) and, thus, contribute to can influence RNA 3′ end processing, often depending on the binding transcriptome diversification (1, 2). Remodeling of 3′ UTRs can have positions within mRNA target 3′ UTRs (4). particularly profound phenotypic consequences; hence, transcript Other protein complexes are involved in the 3′ end processing of isoforms may differ in their relative stability, localization, translation nonpolyadenylated RNA species. For instance, the Integrator com- rate, and/or function (2). Although it is well known that RNA 3′ end plex, which binds to the C-terminal domain (CTD) of the RNA processing can be finely regulated depending on the cellular needs, polymerase II (Pol II), is responsible for the RNA 3′ end processing the factors involved in alternative 3′ end processing are only partially of uridylate-rich small nuclear RNA transcripts (UsnRNAs) (5). This characterized. complex has been shown to control termination of transcription The core of the pre-mRNA 3′ end processing complex consists of and 3′ end processing at enhancer RNAs and replication-dependent four subcomplexes, namely, cleavage and polyadenylation factor (CPSF), histone loci (6, 7). Furthermore, Integrator binding to the proximal cleavage stimulation factor (CSTF), cleavage factor I (CFI), and CFII. promoter region of polyadenylated target genes negatively regulates Few other proteins, including symplekin and polyadenylate [poly(A)] their expression (7). polymerase (PAP), are also involved in completing the 3′ end for- Alternative 3′ end processing–dependent transcriptome diversi- mation of polyadenylated RNA (3). In metazoans, sites of pre-mRNA fication plays key roles in various important biological processes (4). polyadenylation are primarily defined by the canonical poly(A) signal Individual 3′ end processing events have also been implicated under AAUAAA, which is positioned ~21-nucleotides (nt) upstream of the pathological conditions, including autoimmune disorders and cancer cleavage site (3). This hexamer is recognized by the cotranscriptionally (4). Consistent with a reported general association between the ex- recruited CPSF subcomplex, which carries out the endonucleolytic pression of short RNA 3′ UTRs and a proliferative cellular state (8), cleavage event, followed by the addition of a poly(A) tail to the most cancers express transcripts with shorter 3′ UTRs than those 5′ cleavage product by PAP. Deregulation of protein expression levels expressed in corresponding normal tissues (4). Some studies have attributed cancer-related 3′ end RNA patterns to the deregulated activity of specific 3′ end processing factors, such as CSTF2 (9) and 1Laboratory for Molecular Cancer Biology, Center for Cancer Biology, VIB, Leuven, CFIm25 (10). However, the motifs recognized by these core 3′ end 2 Belgium. Laboratory for Molecular Cancer Biology, Department of Oncology, KU processing factors do not explain the observed quantitative changes Leuven, Leuven, Belgium. 3VIB Center for Medical Biotechnology, VIB, 9000 Ghent, Belgium. 4Department of Biomolecular Medicine, Ghent University, 9000 Ghent, in poly(A) site usage between tumor and normal tissue samples from Belgium. 5Laboratory for RNA Cancer Biology, Department of Oncology, LKI, KU The Cancer Genome Atlas (TCGA) (11), indicating that other un- 6 Leuven, Leuven, Belgium. Sylvester Comprehensive Cancer Center, Department of known modulators also contribute. Human Genetics, University of Miami Miller School of Medicine, 1501 NW 10th Avenue, Miami, FL 33136, USA. 7Bioinformatics Core Laboratory, Children’s Hospital Research Deregulation of RNA 3′ end processing at specific loci may also Institute of Manitoba (CHRIM), Winnipeg, Manitoba, Canada. 8Department of contribute to tumor growth as illustrated in recent findings implicating Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada. the long noncoding RNA (lncRNA) locus NEAT1 in cancer devel- 9Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. 10 opment (12). This locus produces two lncRNA isoforms (13). The VIB Proteomics Core, 9000 Ghent, Belgium. *These authors contributed equally to this work. shorter isoform, NEAT1_1 (3700 nt in length), contains a functional †Corresponding author. Email: [email protected] poly(A) site. The long NEAT1_2 isoform (22700 nt in length), which Barra et al., Sci. Adv. 2020; 6 : eaaz9072 3 July 2020 1 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE is not polyadenylated, is produced as a read-through transcript when RAP experiments were performed in biological triplicates, and the 3′ end processing of NEAT1_1 is inefficient (14). The mechanisms purified proteins were analyzed by label-free mass spectrometry underlying NEAT1 isoform switching remain poorly understood. The (MS). Principal component analysis (PCA) of the replicates con- ubiquitous heterogeneous nuclear ribonucleoprotein K (HNRNPK) firmed the clustering of the samples into two groups: the control has been implicated in this process, by competing with CPSF6 for the (Ctrl) and the RAP pulldown performed with NEAT1-specific probes binding of NUDT21 (CFIm25) and impairing NEAT1_1 polyadenylation (N1_5′) (fig. S1A). We identified 34 proteins, which were signifi- (14). Moreover, TAR DNA binding protein 43 (TDP-43) enhances cantly enriched by the N1_5′ probes [t test, P < 0.05 and fold change NEAT1_1 polyadenylation in pluripotent cells (15). Whereas the (FC) > 1.6], as high-confidence NEAT1 interactors (table S1). Two function of NEAT1_1 still needs to be established (16, 17), NEAT1_2 of these were known PS proteins (Fig. 1D) (43). Gene ontology is an essential architectural component of paraspeckles (PS) (18), analysis using the Search Tool for Recurring Instances of Neighboring which are highly ordered and phase-separated nuclear stress bodies Genes (https://string-db.org) indicated that the remaining NEAT1 (19). Thus, PS assembly critically depends on the poorly understood interactors are mainly involved in key aspects of RNA biogenesis NEAT1 isoform switch. Expression of NEAT1_2, and thereby PS and processing (table S2). Among these were multiple subunits of assembly, can only be detected under specific physiological condi- the Integrator and mRNA 3′ end processing complexes, including tions (i.e., lactating mammary glands) and in response to various INTS1, INTS3, INTS6, CSTF1, CSTF2, CSTF2T, CSTF3, CPSF1, forms of stresses, including oncogenic stress (12, 20–22). Accordingly, WDR33, SYMPK, and FIP1L1 (Fig. 1E, top). One-third (11 of 34) PS appear in over 65% of human epithelial cancers (12), where they of all high-confidence interactors belonged to these two multicom- predict poor prognosis (23) and are either completely absent, or only ponent protein complexes. Three additional previously unknown sporadically detectable, in normal tissues (12, 24). In a classical NEAT1 interactors were also identified, namely, the F-box protein two-stage chemically induced skin cancer mouse model, PS are induced FBXO11 and its binding partner CUL1, as well as the transcription in skin epidermal cells exposed to oncogenic stress, while genetic factor TCF7L2 (Fig. 1E, bottom). ablation of NEAT1 markedly