A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma

Circular RNAs (circRNAs) are a large class of transcripts in the mammalian genome. Although the translation of circRNAs was reported, additional coding circRNAs and the functions of their translated products remain elusive. Here, we demonstrate that an endogenous circRNA generated from a long noncoding RNA encodes regulatory peptides. Through ribosome nascent-chain complex-bound RNA sequencing (RNC-seq), we discover several peptides potentially encoded by circRNAs. We identify an 87-amino-acid peptide encoded by the circular form of the long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) that suppresses glioblastoma cell proliferation in vitro and in vivo. This peptide directly interacts with polymerase associated factor complex (PAF1c) and inhibits the transcriptional elongation of multiple oncogenes. The expression of this peptide and its corresponding circRNA are decreased in glioblastoma compared with the levels in normal tissues. Our results establish the existence of peptides encoded by circRNAs and demonstrate their potential functions in glioblastoma tumorigenesis.Functional peptides can be encoded by short open reading frames in non-coding RNA. Here, the authors identify a 87aa peptide encoded by the circular form of the long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) that can reduce glioblastoma proliferation via interaction with PAF1 which sequentially inhibits the transcriptional elongation of some oncogenes.

[1]  Joseph A. Rothnagel,et al.  Emerging evidence for functional peptides encoded by short open reading frames , 2014, Nature Reviews Genetics.

[2]  G. Shan,et al.  Exon-intron circular RNAs regulate transcription in the nucleus , 2015, Nature Structural &Molecular Biology.

[3]  Charles Gawad,et al.  Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types , 2012, PloS one.

[4]  Pierre Tufféry,et al.  PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces , 2014, Nucleic Acids Res..

[5]  Steven J. M. Jones,et al.  Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma , 2016, Cell.

[6]  J. Sikela,et al.  The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis , 2006, Oncogene.

[7]  S. Batra,et al.  PD2/PAF1 at the Crossroads of the Cancer Network. , 2018, Cancer research.

[8]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[9]  N. Rajewsky,et al.  Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis , 2017, Molecular cell.

[10]  B. Strahl,et al.  Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a , 2016, Nature Cell Biology.

[11]  Michael K. Slevin,et al.  Circular RNAs are abundant, conserved, and associated with ALU repeats. , 2013, RNA.

[12]  S. Buratowski,et al.  Leo1 Subunit of the Yeast Paf1 Complex Binds RNA and Contributes to Complex Recruitment* , 2010, The Journal of Biological Chemistry.

[13]  Juan Pablo Couso,et al.  Peptides Encoded by Short ORFs Control Development and Define a New Eukaryotic Gene Family , 2007, PLoS biology.

[14]  Frances M. G. Pearl,et al.  Conserved Regulation of Cardiac Calcium Uptake by Peptides Encoded in Small Open Reading Frames , 2013, Science.

[15]  Michael Morse,et al.  Multiple knockout mouse models reveal lincRNAs are required for life and brain development , 2013, eLife.

[16]  Fuhui Long,et al.  An anatomic transcriptional atlas of human glioblastoma , 2018, Science.

[17]  E. Schuman,et al.  Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity , 2015, Nature Neuroscience.

[18]  N. Rajewsky,et al.  circRNA biogenesis competes with pre-mRNA splicing. , 2014, Molecular cell.

[19]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[20]  Julia Salzman,et al.  Cell-Type Specific Features of Circular RNA Expression , 2013, PLoS genetics.

[21]  Laia Sadeghi,et al.  The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast , 2015, EMBO reports.

[22]  Emily J. Girard,et al.  Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A. , 2013, Genes & development.

[23]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[24]  Tim Schneider,et al.  Exon circularization requires canonical splice signals. , 2015, Cell reports.

[25]  Raffaele Pezzilli,et al.  Common variation at 2 p 13 . 3 , 3 q 29 , 7 p 13 and 17 q 25 . 1 associated with susceptibility to pancreatic cancer , 2022 .

[26]  P. Sarnow,et al.  Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. , 1995, Science.

[27]  Cole Trapnell,et al.  TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions , 2013, Genome Biology.

[28]  Shawn M. Gillespie,et al.  Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma , 2014, Science.

[29]  J. Kieft,et al.  The structures of nonprotein‐coding RNAs that drive internal ribosome entry site function , 2012, Wiley interdisciplinary reviews. RNA.

[30]  Yan Li,et al.  Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs , 2016, Nature Communications.

[31]  N. Rajewsky,et al.  Translation of CircRNAs , 2017, Molecular cell.

[32]  Yang Wang,et al.  Efficient backsplicing produces translatable circular mRNAs , 2015, RNA.

[33]  Joseph A. Rothnagel,et al.  Emerging evidence for functional peptides encoded by short open reading frames , 2014, Nature Reviews Genetics.

[34]  R. Parker,et al.  Circular RNAs: diversity of form and function , 2014, RNA.

[35]  Christophe Dunand,et al.  Primary transcripts of microRNAs encode regulatory peptides , 2015, Nature.

[36]  Kai Wang,et al.  Circular RNA profile in gliomas revealed by identification tool UROBORUS , 2016, Nucleic acids research.

[37]  Ashley R. Woodfin,et al.  PAF1, a Molecular Regulator of Promoter-Proximal Pausing by RNA Polymerase II , 2015, Cell.

[38]  D. Bartel,et al.  Expanded identification and characterization of mammalian circular RNAs , 2014, Genome Biology.

[39]  Ling-Ling Chen,et al.  Complementary Sequence-Mediated Exon Circularization , 2014, Cell.

[40]  S. Gabriel,et al.  Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. , 2010, Cancer cell.

[41]  Daniel G. Anderson,et al.  Engineering circular RNA for potent and stable translation in eukaryotic cells , 2018, Nature Communications.

[42]  P. Pandolfi,et al.  Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations , 2016, Cell.

[43]  Weining Yang,et al.  Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2 , 2016, Nucleic acids research.

[44]  Carmen Birchmeier,et al.  Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function , 2017, Science.

[45]  Liu Ming,et al.  A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis , 2018, Oncogene.

[46]  A. Eyre-Walker,et al.  Hundreds of putatively functional small open reading frames in Drosophila , 2011, Genome Biology.

[47]  M. López-Lastra,et al.  Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation. , 2005, Biological research.

[48]  Yan Li,et al.  circRNADb: A comprehensive database for human circular RNAs with protein-coding annotations , 2016, Scientific Reports.

[49]  N. Sharpless,et al.  Detecting and characterizing circular RNAs , 2014, Nature Biotechnology.

[50]  Jiaying Tan,et al.  The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis. , 2010, Cancer cell.

[51]  Petar Glažar,et al.  circBase: a database for circular RNAs , 2014, RNA.

[52]  Yang Zhang,et al.  Extensive translation of circular RNAs driven by N6-methyladenosine , 2017, Cell Research.

[53]  S. Cherry,et al.  Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins , 2015, Genes & development.

[54]  Ying Chen Eyre-Walker,et al.  Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq , 2014, eLife.

[55]  Qing-Yu He,et al.  Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific , 2013, Nucleic acids research.

[56]  Martin Mokrejs,et al.  IRESite: the database of experimentally verified IRES structures () , 2005, Nucleic Acids Res..

[57]  Petar Glažar,et al.  Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. , 2015, Molecular cell.

[58]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[59]  N. Lemp,et al.  Splicing mediates the activity of four putative cellular internal ribosome entry sites , 2008, Proceedings of the National Academy of Sciences.

[60]  S. Batra,et al.  Human RNA polymerase II-associated factor complex: dysregulation in cancer , 2007, Oncogene.

[61]  Suyun Huang,et al.  Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis , 2017, Journal of the National Cancer Institute.

[62]  Christoph Dieterich,et al.  Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. , 2015, Cell reports.

[63]  J. Rinn,et al.  Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2 , 2013, Genome Biology.