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• W2896075315 abstract "•Differential enrichment of coding and noncoding RNAs in EVs suggests regulated export•The majority of mRNAs in EVs are not full length•Smaller mRNAs (∼1 kb) can be transferred from donor to recipient cells by EVs•lncRNAs can be transferred to recipient cells and trafficked to function in the nucleus The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA. The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA. The majority of the human genome is transcribed into RNA, but only ∼2%–3% encodes protein (Hangauer et al., 2013Hangauer M.J. Vaughn I.W. McManus M.T. Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs.PLoS Genet. 2013; 9: e1003569Crossref PubMed Scopus (525) Google Scholar). Only a small fraction of noncoding RNA transcripts have been characterized, but they appear to play important regulatory roles in multiple biological contexts (Kopp and Mendell, 2018Kopp F. Mendell J.T. Functional classification and experimental dissection of long noncoding RNAs.Cell. 2018; 172: 393-407Abstract Full Text Full Text PDF PubMed Scopus (1917) Google Scholar, Wu et al., 2017Wu H. Yang L. Chen L.L. The diversity of long noncoding RNAs and their generation.Trends Genet. 2017; 33: 540-552Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Recently, numerous studies have demonstrated the presence of distinct types of extracellular RNA (exRNA) in diverse biological fluids, adding yet another surprise to the overall role of RNA in gene expression (Colombo et al., 2014Colombo M. Raposo G. Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.Annu. Rev. Cell Dev. Biol. 2014; 30: 255-289Crossref PubMed Scopus (3627) Google Scholar, Mateescu et al., 2017Mateescu B. Kowal E.J. van Balkom B.W. Bartel S. Bhattacharyya S.N. Buzás E.I. Buck A.H. de Candia P. Chow F.W. Das S. et al.Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.J. Extracell. Vesicles. 2017; 6: 1286095Crossref PubMed Scopus (453) Google Scholar, Tkach and Théry, 2016Tkach M. Théry C. Communication by extracellular vesicles: where we are and where we need to go.Cell. 2016; 164: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (2023) Google Scholar). Because extracellular fluids display abundant ribonuclease activity, exRNA must be protected from degradation in protein complexes (Arroyo et al., 2011Arroyo J.D. Chevillet J.R. Kroh E.M. Ruf I.K. Pritchard C.C. Gibson D.F. Mitchell P.S. Bennett C.F. Pogosova-Agadjanyan E.L. Stirewalt D.L. et al.Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.Proc. Natl. Acad. Sci. USA. 2011; 108: 5003-5008Crossref PubMed Scopus (2509) Google Scholar, Turchinovich et al., 2011Turchinovich A. Weiz L. Langheinz A. Burwinkel B. Characterization of extracellular circulating microRNA.Nucleic Acids Res. 2011; 39: 7223-7233Crossref PubMed Scopus (1475) Google Scholar), lipid complexes (Tabet et al., 2014Tabet F. Vickers K.C. Cuesta Torres L.F. Wiese C.B. Shoucri B.M. Lambert G. Catherinet C. Prado-Lourenco L. Levin M.G. Thacker S. et al.HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells.Nat. Commun. 2014; 5: 3292Crossref PubMed Scopus (298) Google Scholar, Vickers et al., 2011Vickers K.C. Palmisano B.T. Shoucri B.M. Shamburek R.D. Remaley A.T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.Nat. Cell Biol. 2011; 13: 423-433Crossref PubMed Scopus (2112) Google Scholar), or extracellular vesicles (EVs) (Ratajczak et al., 2006Ratajczak J. Miekus K. Kucia M. Zhang J. Reca R. Dvorak P. Ratajczak M.Z. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.Leukemia. 2006; 20: 847-856Crossref PubMed Scopus (1182) Google Scholar, Skog et al., 2008Skog J. Würdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr., W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3720) Google Scholar, Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar). EVs refer to membrane limited nanovesicles including exosomes, microvesicles, and other secreted vesicles (Raposo and Stoorvogel, 2013Raposo G. Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends.J. Cell Biol. 2013; 200: 373-383Crossref PubMed Scopus (5192) Google Scholar). Each class of vesicle is unique in its origin and/or size and thus differs in its composition of lipid, protein, RNA, and potential DNA cargo (Colombo et al., 2014Colombo M. Raposo G. Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.Annu. Rev. Cell Dev. Biol. 2014; 30: 255-289Crossref PubMed Scopus (3627) Google Scholar, Mateescu et al., 2017Mateescu B. Kowal E.J. van Balkom B.W. Bartel S. Bhattacharyya S.N. Buzás E.I. Buck A.H. de Candia P. Chow F.W. Das S. et al.Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.J. Extracell. Vesicles. 2017; 6: 1286095Crossref PubMed Scopus (453) Google Scholar). EVs are released by all cell types and can serve as vehicles for transport of protein and RNA cargo between cells, representing a potential mechanism for intercellular communication (Ratajczak et al., 2006Ratajczak J. Miekus K. Kucia M. Zhang J. Reca R. Dvorak P. Ratajczak M.Z. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.Leukemia. 2006; 20: 847-856Crossref PubMed Scopus (1182) Google Scholar, Skog et al., 2008Skog J. Würdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr., W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3720) Google Scholar, Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar). Local and systemic cargo transfer via EVs has been associated with tumor microenvironment interactions, aggressiveness, and metastasis (Becker et al., 2016Becker A. Thakur B.K. Weiss J.M. Kim H.S. Peinado H. Lyden D. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis.Cancer Cell. 2016; 30: 836-848Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, Kalluri, 2016Kalluri R. The biology and function of exosomes in cancer.J. Clin. Invest. 2016; 126: 1208-1215Crossref PubMed Scopus (1078) Google Scholar, Shurtleff et al., 2018Shurtleff M.J. Temoche-Diaz M.M. Schekman R. Extracellular vesicles and cancer: caveat lector.Annu. Rev. Cancer Biol. 2018; 2: 395-411Crossref Scopus (38) Google Scholar). This potentially allows secretion of proteins and RNAs that could inhibit local growth and simultaneously “educate” distant tissues for metastasis (Peinado et al., 2012Peinado H. Alečković M. Lavotshkin S. Matei I. Costa-Silva B. Moreno-Bueno G. Hergueta-Redondo M. Williams C. García-Santos G. Ghajar C. et al.Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.Nat. Med. 2012; 18: 883-891Crossref PubMed Scopus (2635) Google Scholar). Circulating RNAs encased in vesicles or protein complexes are often altered in cancer and bear tumor-type-specific “signatures,” making them attractive candidates as clinical biomarkers for disease diagnosis and prognosis (Quinn et al., 2015Quinn J.F. Patel T. Wong D. Das S. Freedman J.E. Laurent L.C. Carter B.S. Hochberg F. Van Keuren-Jensen K. Huentelman M. et al.Extracellular RNAs: development as biomarkers of human disease.J. Extracell. Vesicles. 2015; 4: 27495Crossref PubMed Scopus (58) Google Scholar). Many exRNA studies have focused on miRNAs because they are well characterized, small, relatively stable, and well annotated (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar, Mittelbrunn et al., 2011Mittelbrunn M. Gutiérrez-Vázquez C. Villarroya-Beltri C. González S. Sánchez-Cabo F. González M.A. Bernad A. Sánchez-Madrid F. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.Nat. Commun. 2011; 2: 282Crossref PubMed Scopus (1320) Google Scholar, Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar, Vickers et al., 2011Vickers K.C. Palmisano B.T. Shoucri B.M. Shamburek R.D. Remaley A.T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.Nat. Cell Biol. 2011; 13: 423-433Crossref PubMed Scopus (2112) Google Scholar). However, the diversity of exRNA is extensive and microRNAs (miRNAs) are not the most abundant class of RNA found in EVs (Fritz et al., 2016Fritz J.V. Heintz-Buschart A. Ghosal A. Wampach L. Etheridge A. Galas D. Wilmes P. Sources and functions of extracellular small RNAs in human circulation.Annu. Rev. Nutr. 2016; 36: 301-336Crossref PubMed Scopus (85) Google Scholar, Mateescu et al., 2017Mateescu B. Kowal E.J. van Balkom B.W. Bartel S. Bhattacharyya S.N. Buzás E.I. Buck A.H. de Candia P. Chow F.W. Das S. et al.Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.J. Extracell. Vesicles. 2017; 6: 1286095Crossref PubMed Scopus (453) Google Scholar). Analysis of cellular versus exRNA has repeatedly demonstrated selective biogenesis, export, and/or stability of specific RNAs (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar, Dou et al., 2016Dou Y. Cha D.J. Franklin J.L. Higginbotham J.N. Jeppesen D.K. Weaver A.M. Prasad N. Levy S. Coffey R.J. Patton J.G. Zhang B. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.Sci. Rep. 2016; 6: 37982Crossref PubMed Scopus (246) Google Scholar, Kosaka et al., 2010Kosaka N. Iguchi H. Yoshioka Y. Takeshita F. Matsuki Y. Ochiya T. Secretory mechanisms and intercellular transfer of microRNAs in living cells.J. Biol. Chem. 2010; 285: 17442-17452Crossref PubMed Scopus (1512) Google Scholar, Santangelo et al., 2016Santangelo L. Giurato G. Cicchini C. Montaldo C. Mancone C. Tarallo R. Battistelli C. Alonzi T. Weisz A. Tripodi M. The RNA-binding protein SYNCRIP is a component of the hepatocyte exosomal machinery controlling microRNA sorting.Cell Rep. 2016; 17: 799-808Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, Skog et al., 2008Skog J. Würdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr., W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3720) Google Scholar, Squadrito et al., 2014Squadrito M.L. Baer C. Burdet F. Maderna C. Gilfillan G.D. Lyle R. Ibberson M. De Palma M. Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells.Cell Rep. 2014; 8: 1432-1446Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, Valadi et al., 2007Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar, Villarroya-Beltri et al., 2013Villarroya-Beltri C. Gutiérrez-Vázquez C. Sánchez-Cabo F. Pérez-Hernández D. Vázquez J. Martin-Cofreces N. Martinez-Herrera D.J. Pascual-Montano A. Mittelbrunn M. Sánchez-Madrid F. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs.Nat. Commun. 2013; 4: 2980Crossref PubMed Scopus (1237) Google Scholar, Wei et al., 2017Wei Z. Batagov A.O. Schinelli S. Wang J. Wang Y. El Fatimy R. Rabinovsky R. Balaj L. Chen C.C. Hochberg F. et al.Coding and noncoding landscape of extracellular RNA released by human glioma stem cells.Nat. Commun. 2017; 8: 1145Crossref PubMed Scopus (291) Google Scholar). Elucidation of the mechanisms for selective sorting of cargo into EVs is critical to understanding extracellular signaling by RNA. In our ongoing efforts to understand the biological and pathological role of exRNAs regulated by oncogenic signaling, we utilized three isogenic colorectal cancer (CRC) cell lines that differ only in the mutational status of the KRAS gene (Shirasawa et al., 1993Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.Science. 1993; 260: 85-88Crossref PubMed Scopus (601) Google Scholar). KRAS mutations occur in ∼34%–45% of colon cancers (Wong and Cunningham, 2008Wong R. Cunningham D. Using predictive biomarkers to select patients with advanced colorectal cancer for treatment with epidermal growth factor receptor antibodies.J. Clin. Oncol. 2008; 26: 5668-5670Crossref PubMed Scopus (81) Google Scholar). The parental DLD-1 cell line contains both WT and G13D mutant KRAS alleles, while the isogenically matched derivative cell lines contain only one mutant KRAS allele (DKO-1) or one WT KRAS allele (DKs-8) (Shirasawa et al., 1993Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.Science. 1993; 260: 85-88Crossref PubMed Scopus (601) Google Scholar). We previously showed that EVs from mutant KRAS CRC cells can be transferred to WT cells to induce cell growth, migration, and invasiveness (Demory Beckler et al., 2013Demory Beckler M. Higginbotham J.N. Franklin J.L. Ham A.J. Halvey P.J. Imasuen I.E. Whitwell C. Li M. Liebler D.C. Coffey R.J. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.Mol. Cell. Proteomics. 2013; 12: 343-355Crossref PubMed Scopus (391) Google Scholar, Higginbotham et al., 2011Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Additionally, we found that the miRNA profiles of EVs from all three cell lines are distinct from the parental cells and segregate depending on KRAS status and that specific miRNAs can be functionally transferred from mutant KRAS cells to WT cells (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar). We also found that specific intracellular oncogenic signaling events can regulate trafficking of miRNAs through phosphorylation of Argonaute (AGO) proteins (McKenzie et al., 2016McKenzie A.J. Hoshino D. Hong N.H. Cha D.J. Franklin J.L. Coffey R.J. Patton J.G. Weaver A.M. KRAS-MEK signaling controls Ago2 and miRNA sorting into exosomes.Cell Rep. 2016; 15: 978-987Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). More recently, we identified a global downregulation of circular RNAs (circRNAs) in mutant KRAS cells with an inverse upregulation in EVs (Dou et al., 2016Dou Y. Cha D.J. Franklin J.L. Higginbotham J.N. Jeppesen D.K. Weaver A.M. Prasad N. Levy S. Coffey R.J. Patton J.G. Zhang B. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.Sci. Rep. 2016; 6: 37982Crossref PubMed Scopus (246) Google Scholar). Here, we report comprehensive analysis of EV long RNAs (>200 nt) and show that similar to miRNA export, there is selective export of long RNAs to EVs. We also show that both mRNAs and long noncoding RNAs (lncRNAs) can be functionally transferred between cells. We previously isolated EVs through a series of differential ultracentrifugation steps and showed that the vesicles were ∼40–130 nm in diameter with the typical cup-shaped electron microscopy (EM) morphology and the expected pattern of protein markers consistent with endosome derived exosomes (Demory Beckler et al., 2013Demory Beckler M. Higginbotham J.N. Franklin J.L. Ham A.J. Halvey P.J. Imasuen I.E. Whitwell C. Li M. Liebler D.C. Coffey R.J. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.Mol. Cell. Proteomics. 2013; 12: 343-355Crossref PubMed Scopus (391) Google Scholar, Higginbotham et al., 2011Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Protein and RNA were isolated from these vesicles for proteomics, small RNA sequencing (RNA-seq), and circular RNA analysis (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar, Demory Beckler et al., 2013Demory Beckler M. Higginbotham J.N. Franklin J.L. Ham A.J. Halvey P.J. Imasuen I.E. Whitwell C. Li M. Liebler D.C. Coffey R.J. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.Mol. Cell. Proteomics. 2013; 12: 343-355Crossref PubMed Scopus (391) Google Scholar, Dou et al., 2016Dou Y. Cha D.J. Franklin J.L. Higginbotham J.N. Jeppesen D.K. Weaver A.M. Prasad N. Levy S. Coffey R.J. Patton J.G. Zhang B. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.Sci. Rep. 2016; 6: 37982Crossref PubMed Scopus (246) Google Scholar). In all cases, we found that EVs derived from mutant KRAS cell lines have distinct proteomes, miRNA profiles, and circular RNA profiles compared to their parental cellular patterns and EVs from WT KRAS cells. Here, we analyzed long RNA-seq libraries generated from the same vesicle and RNA preparations to determine whether long RNAs are selectively sorted into EVs from CRC cells. Long RNA-seq was performed on rRNA-depleted total cellular RNA, and the cellular RNA profiles were then compared to EV RNA profiles. Without rRNA depletion, the majority of RNA-seq reads were derived from rRNA, whereas depletion allowed for more ready comparison of other differentially enriched RNAs. Comparison of RNA-seq libraries with or without rRNA depletion showed no significant effect on the detection of up- or downregulated long RNA reads (data not shown). In our previous papers, we referred to our vesicle preparations as exosomes, but we are aware that our preparations contain mixtures of lipoproteins and other protein complexes. Moving forward, we will refer to these vesicles as EVs. Regardless of nomenclature, all of our analyses are derived from the same preparations, allowing direct comparison to previous proteomic, small RNA-seq, and circular RNA-seq data (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar, Demory Beckler et al., 2013Demory Beckler M. Higginbotham J.N. Franklin J.L. Ham A.J. Halvey P.J. Imasuen I.E. Whitwell C. Li M. Liebler D.C. Coffey R.J. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.Mol. Cell. Proteomics. 2013; 12: 343-355Crossref PubMed Scopus (391) Google Scholar, Dou et al., 2016Dou Y. Cha D.J. Franklin J.L. Higginbotham J.N. Jeppesen D.K. Weaver A.M. Prasad N. Levy S. Coffey R.J. Patton J.G. Zhang B. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.Sci. Rep. 2016; 6: 37982Crossref PubMed Scopus (246) Google Scholar). As a first analysis of the long RNA-seq data, we mapped reads to the human genome against both annotated and unannotated regions, allowing a maximum of 2 total mismatches. Alignment in this manner revealed that the mapping percentages were higher in cellular datasets than EV datasets. For all cellular profiles, ∼70% of paired reads mapped to unique sequences in the human genome, while in EVs, the mapping percentages were much lower (30%–50%) (Figure 1A). The decreased abundance of unique mappable reads in the EV libraries was not due to amplification and sequencing of contaminating RNAs but rather because multiple mismatches were more prevalent in EV RNA than in parental cellular RNA. One possible explanation for the increase in mismatched reads is the presence of substantially more modified RNAs in EVs, which could alter base incorporation during library construction (unpublished data). We next restricted read mapping to unambiguous annotated genomic regions and then performed pairwise analyses between samples. In this case, the cell replicates showed high correlation (r = 0. 91–0.94), while the EV replicates showed more variation between triplicate samples (r = 0.76–0.95) (Figure S1). We did not observe as much variability when analyzing extracellular small RNAs or for our proteomic analyses, but for unknown reasons (possibly batch effects), we observed increased variability when preparing long RNA libraries. The variability was not due to differential quality of the input RNA or lower sequencing depth. Nevertheless, when we compared the RNA profiles from EVs to their parental cells and focused only on annotated genes, the analysis showed that specific RNAs are selectively exported into EVs, consistent with low correlation between EVs and cells (DKO-1 r = 0.68–0.72, DKs-8 r = 0.68–0.78, DLD-1 r = 0.66–0.73) (Figure S1). In the cellular datasets, a majority of reads corresponded to unique annotated gene regions (∼60%), while a much smaller percentage of reads in the EV profiles mapped to unique annotated genes (∼5%–15%) (Figure 1B). The remaining unassigned reads in both the cellular and EV datasets represent RNA species that map to unannotated loci. This is supported by the difference in the percentage of unique mapped reads in both the cellular (∼70%) and EV samples (∼30%–50%) when mapping was not restricted to annotated gene regions (Figure 1A). Although the majority of long RNA-seq reads mapped to known protein coding sequences in both the cellular and EV datasets, we discovered differential enrichment of specific RNAs between cells and EVs (Figure 1C). In cells, a higher percentage of transcripts corresponded to protein coding genes and known lncRNAs as compared to EVs. For EVs, we observed enrichment of transcripts derived from pseudogenes and antisense RNAs, with pseudogene transcripts being almost undetectable in cellular samples (≤0.3%) (Figure 1C). To better approximate the expression levels of RNAs as opposed to individual read counts, RNAs were plotted by subtype normalized to RPKM (per kilobase million). As expected with such normalization, smaller RNAs became more prevalent in both the cellular and extracellular samples, but the relative differential enrichment was unaffected (Figure 1D). To further analyze whether the overall long RNA profiles are distinct between cells and EVs and between WT and mutant KRAS, we performed principal-component analysis (PCA). PCA revealed that the repertoire of long RNAs is clearly distinct when comparing parental cellular RNA profiles and their secreted EV RNAs (Figure 2A). Across the three cell lines, RNA expression patterns clustered together and were distinct from EVs, indicating that KRAS-driven differential RNA expression is less pronounced when comparing patterns across cells than when comparing patterns between EVs and their cognate cell lines. However, if the cellular RNA expression patterns are compared alone, then the profiles segregate by KRAS status under a variety of culture conditions (Figure S2). Differential gene expression analyses were performed comparing cellular RNAs to their cognate EVs, comparing cellular RNAs among the three cell lines differing in KRAS status (mutant cell/WT cell) and comparing EV RNA profiles differing in KRAS status (mutant EV/WT EV). The top differentially expressed RNAs in either WT or mutant KRAS cells and exosomes are shown in Table 1. When comparing EVs to their parent cells, we found that DKO-1 mutant KRAS EVs were enriched for a dramatically different population of RNAs compared to EVs from either WT (DKs-8) or heterozygote (DLD-1) KRAS EVs (Figure 2B). A similar trend was observed when comparing cellular RNA patterns in mutant versus WT or heterozygote cell lines (Figure 2C). Thus, the diversity of RNAs targeted for export is much greater in mutant KRAS cells, especially in cells with a single mutant allele and no WT KRAS allele (DKO-1). It is important to note that the vast majority of long RNAs upregulated in EVs are shared across all three isogenic lines (12,814 RNAs), suggesting export mechanisms for these RNAs are largely KRAS independent (Figure 2B). This is in contrast to what we observed in previous miRNA profiles, where the majority of RNAs were upregulated in DKO-1 EVs (Cha et al., 2015Cha D.J. Franklin J.L. Dou Y. Liu Q. Higginbotham J.N. Demory Beckler M. Weaver A.M. Vickers K. Prasad N. Levy S. et al.KRAS-dependent sorting of miRNA to exosomes.eLife. 2015; 4: e07197Crossref PubMed Scopus (234) Google Scholar). Proteomic profiles from DKO-1- and DLD-1-derived EVs were more similar to each other when compared to Dks-8-derived EVs (Demory Beckler et al., 2013Demory Beckler M. Higginbotham J.N. Franklin J.L. Ham A.J. Halvey P.J. Imasuen I.E. Whitwell C. Li M. Liebler D.C. Coffey R.J. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.Mol. Cell. Proteomics. 2013; 12: 343-355Crossref PubMed Scopus (391) Google Scholar).Table 1Top 10 Coding and Noncoding Long RNAs Enriched in EVs and KRAS Mutant and WT CellsGeneMutant KRAS EV Enrichment RNA SubtypeFCGeneWT KRAS EV Enrichment RNA SubtypeFCNoncodingCTD-2328D6.1lincRNA11.43CTD-2328D6.1lincRNA11.67REXO1L1PPseudogene9.05REXO1L1PREXO1-like 1, pseudogene9.30ERVH-1endogenous retrovirus group H member 18.47LINC01609lincRNA7.69LINC01609lincRNA8.21ESRGembryonic stem cell related (non-protein coding)7.15ESRGembryonic stem cell related (non-protein coding)7.98LINC00504lincRNA7.08LINC00504Antisense RNA7.34AL590867.1lincRNA7.06LINC02503lincRNA7.15LINC02503lincRNA6.70AC087473.1lincRNA7.07LACTB2-AS1LACTB2 antisense RNA 16.47AL671511.1lincRNA6.96TPTEP1TPTE pseudogene 16.28UGDH-AS1UGDH a" @default.
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• W2896075315 date "2018-10-01" @default.
• W2896075315 modified "2023-10-07" @default.
• W2896075315 title "Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells" @default.
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• W2896075315 doi "https://doi.org/10.1016/j.celrep.2018.09.054" @default.
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