Alterations in primary RNA motifs and aberrant expression levels of non-coding RNA molecules have emerged as biomarkers of disease development and progression. Advances in antisense oligonucleotide (ASO) techniques and pharmacologic discoveries in targeting of RNA structures and RNA–protein interactions with small molecules open a new area in RNA therapeutics that may help in developing a next generation of anti-cancer drugs.cis-acting RNA motifs have emerged as biomarkers of disease development and progression, opening avenues for the design of novel therapeutic approaches. This review looks at the progress, challenges and future prospects of targeting cis-acting and trans-acting RNA elements for leukemia diagnosis and treatment.RNA molecules are a source of phenotypic diversity and an operating system that connects multiple genetic and metabolic processes in the cell. A dysregulated RNA network is a common feature of cancer. Aberrant expression of long non-coding RNA (lncRNA), micro RNA (miRNA), and circular RNA (circRNA) in tumors compared to their normal counterparts, as well as the recurrent mutations in functional regulatory  Leukemia, a wide spectrum of blood cancers displaying abnormal proliferation and differentiation capacity of myeloid or lymphoid blood progenitors, is the most frequent type of cancer in children and one of the most common in adults . Acute mThe genetics of adult and pediatric leukemia have been intensively studied ,7,8,9. SThe updated human reference genome, GRCh38.p13, contains 2.95 Gb of sequence, approximately 98% of which does not code for proteins ,15.cis-acting elements of RNA influence transcripts’ fate internally, while trans-acting RNA regulatory factors function through binding with proteins, DNA, or other RNA. Therefore, transcriptomes, a collection of all RNA sequences transcribed from the genomic code, capture gene expression complexity beyond a simple reproduction of a nucleic acid order [Genes are generally defined as a fragment of chromosomal DNA that is transcribed into a functional RNA molecule or into RNA translated into a functional protein. As such, the total number of human genes is in the range of 40,000 to 50,000 genetic units . Howeverid order .trans play an important role in neoplasm development and progression [Attempts to catalogue genomic variations across the human population brought to light large-scale structural variants in human genomes which were previously disregarded ,19. Thougression ,24.cis-acting motifs and regulatory ncRNA such as lncRNA, circRNA, and miRNA in blood cancer, giving special attention to pediatric tumors. We discuss how alterations in primary RNA structure and expression levels of regulatory RNA molecules may serve as leukemia biomarkers. Finally, we look at current approaches for chemical and antisense oligonucleotide (ASO) RNA targeting.Here, we overview the role of cis-acting regulatory motif is a primary RNA sequence, often folded into a distinctive secondary structure , internal ribosome entry site (IRES)) that regulates fate and activity of RNA itself through interaction with other RNA molecules, DNA, or RNA-binding proteins (RBPs). When located in introns of messenger RNA precursors (pre-mRNA) and untranslated regions (UTRs) of mature mRNA, these regulatory sequences influence pre-mRNA processing, mRNA stability, translation, transport, and decay. The structural integrity of cis- and trans-acting elements defines the strength and physiological outcomes of their interactions, presenting an additional layer of gene expression control. Alterations in cis-acting regulatory RNA motifs are often detected in the context of cancer and other diseases.RNA cis-regulatory pre-mRNA motifs at the 5′ splice site (GT), the polypyrimidine tract (PPT), the 3′ splice site (AG), and the branch point sequence (BPS). Auxiliary or supplemental splice regulatory elements known as exonic and intronic splicing enhancers and silencers are defined by their effects on adjacent splice sites by either promoting or inhibiting exon inclusion [Pre-mRNAs are primary transcripts consisting of exons (protein-coding regions with an average size of about 200 base pairs) and introns (much lengthier non-coding sequences flanking exons). Splicing, a process of intron removal and connection of exons by a mega-Daltons spliceosomal complex , producenclusion .Somatic or hereditary mutations at the exon-intron boundaries and splicing regulatory motifs are the most common types of mutations leading to mRNA mis-splicing. β-thalassemia, a hereditary blood disorder characterized by anemia and a variety of growth and metabolic abnormalities, was one of the first discovered diseases with splicing site mutations negatively affecting production of functional protein (hemoglobin) . FurtherRelapsed pediatric B-ALL can present with both mutations in protein coding sequences and aberrant splicing of CD19 mRNA. To investigate the molecular nature of resistance to CAR-19 therapies, Thomas-Tikhonenko’s group compared whole exome and RNA sequencing analysis of CART-19 relapse to the CD19-positive, pre-CART-19 leukemia samples from the same patients . ResearcGenetic studies of leukemia and other types of cancer indicate that splice site mutations span multiple loci in pre-mRNAs and often coincide with aberrations in protein-coding sequences of the gene. This is unlike hereditary disorders caused by a single mutation or changes in several base pairs  that can be treated with antisense oligonucleotides. In addition, the molecular mechanisms of mis-splicing in cancer are not limited to mutations in pre-mRNA sequences but involve dysfunction of protein and RNA components of the spliceosome . For exaMutations in splicing factor genes are especially common for adult chronic myeloid and lymphoblastic leukemia . ConversHigh tissue- or context-specificity is another important characteristic of differential splicing in normal and malignant hematopoietic tissues. The analysis of alternatively expressed isoforms between aging hematopoietic stem cells (HSCs) and progenitor cells (HPCs) identified a significant divergence with only few isoforms of transcription and histone regulators being commonly upregulated . Rojas ecis-acting elements and distinctive secondary structures (hairpins) that are recognized by regulatory ncRNA and RBPs. Similar to splicing, recurrent UTRs abnormalities were found in cancer and previously reviewed [The untranslated regions in mRNA (5′ UTR and 3′ UTR) originate from pre-mRNA exons and flank a protein-coding sequence of mature messenger RNA on both sides of an open reading frame (ORF). The UTRs are rich in reviewed ,40,41.GATA2 gene, including 5′UTR, that cumulatively lead to GATA2 loss-of-function [Alterations in 5′ UTRs can disrupt both translation efficiency and protein characteristics. For example, mutations in the 5′ UTR of ANKRD26, the Ankirin Repeat Domain 26 Gene, lead to expression of N-terminally truncated protein and cause the autosomal-dominant form of inherited thrombocytopenia and increase predisposition to AML ,43. The function .With the right sequence context in translation initiation sites (TIS), certain non-AUG start codons can generate expression comparable to a canonical, AUG start codon, whereas mutations in TIS change levels of expression . EndogenNPM1) exhibited increased ribosome occupancy in upstream CUG rather than in conventional AUG initiation sites of canonical ORFs. In addition to a selective generation of oncogenic proteins through unconventional start codons, researchers found a shift of transcriptome towards pathways of stemness and mediators of Wnt/β-catenin signaling [In the context of stress-related global repression of translation, the production of certain oncogenic proteins can increase due to the stress-induced activation of previously repressed upstream start codons . Sendoelignaling ,52. ThesAlternative cleavage and polyadenylation (APA) are a differential selection of AAUAAA polyadenylation sites in 3ʹUTR by APA factors, leading to the expression of different mRNA isoforms that code for the same protein ,54. APA A meta-data analysis of microarray data by Mayr and Bartel demonstrated that shorter mRNA isoforms in cancer cells display increased stability through the loss of microRNA-mediated repression and typically produce ten-fold more protein . The bioMLL) gene that lack its native 3′ UTR are associated with the increased activity of those fusions in leukemia cell lines and tumors compared to fusions that retain MLL 3′ UTR [Cyclin D1 mRNA levels, expressing short Cyclin D1 mRNA isoforms with truncated 3′ UTRs [3′ UTR shortening is associated with increased activity of oncogenes in blood and immune cells. For example, fusion transcripts of the Mixed Lineage Leukemia , the successfully processed and translated messengers can produce atypical, tumor associated neopeptides. As discussed above, alteration in 5′ UTRs of mRNAs can also increase the production of cancer-specific protein isoforms from non-canonical TIS. Hematologic malignances, especially AML, often reveal antigens not expressed by normal cells. That leukemia associated antigens are targeted by αβ and γδ T cells, NKT and NK cells that are proven to be functional against AML in combination with effector ligands and cytokines  ,72. If pTP53 and GATA3 genes [The large whole exome sequencing (WES) and RNA-seq studies identified widespread splicing alterations in around 30% of differentially expressed transcripts. Even though many of them are not cancer drivers, those aberrations can contribute to tumor immunogenicity . JayasinA3 genes . Tumors A3 genes .Trans-acting ncRNAs regulate gene expression in distal genomic regions while cis-acting RNA molecules attenuate gene expression of the locus of their origin or nearby .Several large-scale and single cell sequencing studies explored transcriptomes of normal and malignant hematopoietic cells ,80,81,82Research strategies elucidating the role of ncRNAs in leukemia can be summarized as follows: (i) identification of highly up- or downregulated ncRNA common for certain histological and cytogenetic subtypes of leukemia by analyzing either primary tumors and body fluids, or previously published arrays such as The Cancer Genome Atlas (TCGA) database; (ii) evaluating ncRNAs as potential biomarkers of leukemia in a relationship with white cell blood count, overall survival (OS), event- or disease-free survival , minimal residual disease (MRD), and risk of relapse; (iii) mechanistic studies of ncRNA function in a cell through interaction with DNA, RNA, and protein targets. Finally, a large body of work has been dedicated to understanding the role of ncRNA in chemoresistance and developing anti-ncRNA targeted therapies.CYRANO, MALAT1, NEAT1 and MEG3, are significantly enriched in protein–RNA interaction motifs [Long non-coding RNAs are primary RNA transcripts over 200 nucleotides in length, which are named and categorized based on their genomic origin. Relative to protein-coding sequences, lncRNAs are defined as (i) sense-overlapping, antisense-overlapping, or both (ii) bi-directional, transcribed from sense and anti-sense DNA strands of neighbor genes; (iii) intronic, when transcribed from distal introns; and (iv) intervening/intergenic (lincRNA), not overlapping with annotated coding genes ,84. The n motifs . The spen motifs .Through binding with DNA, RNA, and proteins in the nucleus and cytoplasm, lncRNAs influence gene expression epigenetically, co-transcriptionally, and post-transcriptionally, acting as oncogenes ,92,93,94X-inactive specific transcript (XIST) is a large, 17 kb, transcript involved in X-chromosome genes’ inactivation. Several conserved repeats of XIST mediate recruitment of the epigenetic Polycomb Repressive Complexes (PRC), initiating gene silencing on X chromosome [Xist in the murine blood compartment induced highly aggressive MDS/MPN suggesting that Xist has a genome-wide impact and acts as a potent suppressor of myeloid blood malignancies [One of well-studied lncRNAs, romosome . Deletiognancies .HOX gene loci-associated cis-acting lncRNAs, HOX transcript antisense RNA (HOTAIR) and HOXA transcript at the distal tip (HOTTIP), program active chromatin through interaction with Polycomb and other adapter proteins and play oncogenic roles in leukemia [HOTTIP in AML and showed that HOTTIP coordinates HOXA-driven topologically associated domain (TAD), including the expression of the posterior HOXA genes. HOTTIP also binds in trans with promoters of key hematopoietic regulators like PBX3, MYC, KIT, CD33, MEIS2, and RUNX1. In mice, Hottip displayed oncogenic properties leading to AML-like disease by altering the homeotic-hematopoietic gene-associated chromatin signature and transcription programs [leukemia ,109. Recprograms ,101.HOTAIR sustains leukemia growth and proliferation by negative epigenetic regulation of p15 genes in the nucleus and by sponging miR-193a away from c-KIT mRNA in the cytoplasm [trans-acting lncRNA enhancing oncogene expression through miRNA titration, or a competing endogenous RNA (ceRNA), is CCAT1. Often upregulated in M4-M5 subtypes of AML, CCAT1 inhibits monocytic differentiation and promotes proliferation by reducing miR-155 availability and consequently increases levels of c-MYC [Oncogenic lncRNA ytoplasm ,111. Anoof c-MYC .HOTAIRM1, located in HOXA cluster, is a tumor suppressor regulating selective induction of HOXA1, HOXA4, and myeloid markers CD11b, CD18, and CD11c in NB-4 human acute promyeloblastic leukemia [HOTAIRM1 expression is associated with myeloid lineage specification and ATRA-driven cell cycle arrest. Another anti-leukemic mechanism of HOTAIRM1 action implements degradation of PML-RARA oncoprotein and support an autophagy pathway by withdrawing miR-20a, miR-106b, and miR-125b from ULK1, E2F1, and DRAM2 mRNAs [The intergenic lncRNA leukemia ,114,115.M2 mRNAs .PU.1 sense and antisense RNAs, regulated from shared cis-regulatory DNA elements, is important for maintaining physiological dosage of PU.1 [PU.1 anti-sense transcript (PU.1-AS) disrupts PU.1 translation between the initiation and elongation steps by selective binding with eIF4A initiation factor [PU.1-AS leads to downregulation of PU.1 and promotes myeloid leukemia [AS-RBM15, an anti-sense RNA transcribed in the opposite direction within exon 1 of the megakaryocytic regulator RBM15, promotes terminal differentiation of hematopoietic progenitors by enhancing RBM15 translation in a 5′ cap-dependent manner. The overlapping region between AS-RBM15 RNA and 5′ UTR of RBM15 mRNA functions as an enhancer of RBM15 protein synthesis in megakaryocytic leukemia [In addition to miRNA sponging, lncRNA are capable of altering protein synthesis by interfering with translational machinery. Daniel Tenen’s group showed that the interplay between  of PU.1 . Originan factor . Therefoleukemia . Conversleukemia .FLT3-ITD, NPM1, CEBPA, IGD2, and RUNX1 mutations [NPM1, CEBPA, and FLT3-ITD mutations displayed differential lncRNA expression [XLOC_109948, was identified by Etienne De Clara et al. in the large-scale bioinformatic analysis of NPM1-mutated AML [XLOC_109948 were associated with good treatment outcomes. Downregulation of XLOC_109948 in a NPM1-mutated OCI-AML3 cell line treated with Ara-C or ATRA enhanced apoptosis, thus suggesting the role of this lncRNA in drug sensitivity [To evaluate the prognostic significance of differentially expressed lncRNA in the genetically diverse AML, the de novo RNA sequenced bone marrow samples or TCGA data were thoroughly investigated by several groups. Garzon et al. identified a small subset of lncRNAs strongly correlated with the treatment response and survival of elderly patients (>60-year-old) with cytogenetically normal, untreated AML harboring utations . The folpression . A novelated AML . Low expsitivity .UCA1) lncRNA was upregulated in some pediatric AML after adriamycin (ADR)-based chemotherapy [UCA1 increased the cytotoxic effect of ADR and inhibited HIF-1α-dependent glycolysis in ADR-resistant AML. Mechanistically, UCA1 positively regulates hexokinase 2 and HIF-1α-dependent glycolysis in HL-60 APL by sponging miRNA-125a. In the ADR resistant T-ALL, antisense CDKN2B-AS1 positively regulates TRAF5 by sponging miR-335-3p [lncRNAs contribute to proliferation ,125,126,otherapy . KnockdoR-335-3p .TEL-AML1; t  E2A-PBX1; and t  MLL-AF4) identified most differentially regulated lncRNAs  [BALR-2) and B-ALL associated long RNA-6 (BALR-6) are linked with shorter overall survival, while their inhibition decreases cell proliferation and induces apoptosis [BALR-2 inhibits the glucocorticoid receptor signaling pathway, while BALR-6 negatively regulates activity of SP1(PU.1) and its downstream targets.The molecular analysis of different subsets of pediatric B-ALL , NC00958) . High expoptosis ,135. TheSubtype-specific ncRNA, including lncRNA, in the six major subgroups of pediatric AML , t , t , t , acute megakaryoblastic leukemia (AMKL), and Down syndrome myeloid leukemia (ML-DS)) were described by Schwarzer et al. . The resPCAT7, PVT1, and HOTAIR among the most commonly expressed lncRNAs. The somatic copy number alterations (SCNA) via SNP microarray showed that ovarian and lung cancers had the most of high-frequency (>25%) loss- or gain-of-function lncRNA SCNAs. Whereas AML displayed very few SCNAs, high expression of Breast Cancer Associated lncRNA8 (BCAL8) correlated with poor prognosis. Cancer-associated index SNPs were located in 11.7% of lncRNA loci, and roughly half of them were found in close proximity to protein-coding genes.A comprehensive genomic study of 5037 tumor samples and 935 cancer cell lines among 13 types of cancer, including leukemia, revealed both commonly expressed and cancer-type specific lncRNAs . CompareGao et al. analyzed the impact of somatic mutations and lncRNA expression across 17 cancer types, and its connection with miRNA expression, methylation, and TF-lncRNA interaction . The sciCircular RNAs (circRNAs) are single-stranded RNA sequences covalently linked into circles that range from 100 nt to over 4 kb in size. Similar to miRNA and lncRNA, they comprise evolutionary conserved genomic regions. The biogenesis of circRNAs is linked to splicing and circularization, so-called back-splicing, of exonic, intronic, and other non-coding fragments of newly transcribed RNA. Similar to lncRNA, circRNAs can be translated to proteins and negatively regulate miRNA function by competing with their RNA targets.The stable structure of circRNAs suggests a long-lasting effect on cellular physiology, making circRNAs suitable diagnostic and prognostic markers. In fact, circRNAs, most intensively studied in AML, were identified as potential biomarkers that can be applied at diagnosis, remission, or associated with resistance to therapy .hsa_circ_0004277 expression with AML development: hsa_circ_0004277 levels were significantly downregulated at diagnosis and normalized in remission [circ-ANAPC7 was also proposed to be an additional marker to identify AML [Vimentin gene (circ-VIM) is significantly upregulated and associated with shorter survival in patients with non-acute promyelocytic leukemia and cytogenetically normal AML [circPAN3 was shown to contribute to drug resistance through the circPAN3-miR-153-5p/miR-183-5p-XIAP axis [circ-PVT1. Similar to lncRNA-PVT1, one of the most common long non-coding RNAs, circ-PVT1 is upregulated in AML and ALL and promotes cell proliferation through supporting c-MYC expression by sponging let-7 family and miR-125 [For example, circ-RNA microarray screening of 115 human samples revealed a strong association of emission . circ-ANtify AML , but furrmal AML . High lermal AML . Though rmal AML . circPANIAP axis . Another miR-125 ,149.circ-0004136 acts as a sponge for several pediatric AML-related miRNAs. The bioinformatic algorithm indicated that target genes involved in the circ0004136-miRNA-mRNA network are enriched in leukemia-related signaling pathways. Circ-0004136 expression was significantly upregulated in pediatric AML and potentially sponged AML-related miRNAs, such as miR-29a and miR-142 [MLL fusion partner AF4, circAF4, and other oncogenes  [Analysis of 12 pediatric AML and healthy controls identified 273 upregulated and 296 downregulated circRNAs, mostly transcribed from chromosomes 1, 6, and 16. Among 20 highly upregulated circRNAs,  miR-142 . Studiesd HIPK3) ,152.Cis-acting RNA motifs determine biogenesis and functions of circRNAs [cis-acting elements can potentially abolish or increase expression of circRNAs. The genome-wide in silico search for genetic variants of human circRNAs and analysis of cancer datasets showed that chromosome 17 has a relatively large number of health-related genetic circRNA variants, chromosome 7 contains the highest number of complex mutations, and chromosomes 2 and 1 exhibited the highest number of cancer-related variants. The circRNA-related genetic SNPs, insertions and deletions (INDEL) that might be common for multiple circRNAs have not yet been reported [circRNAs . Typicalreported .The small and medium size, 18-200 nt, non-coding RNAs, e.g., small interfering RNAs (siRNAs), micro RNAs (miRNAs), PIWI-interacting RNAs (piRNAs), small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), promoter-associated small RNAs (PASRs), transcription initiation RNAs (tiRNAs), telomere small RNAs (tel-sRNAs), centrosome-associated RNAs (crasiRNAs), and many others, compose an array of endogenous molecules regulating multiple processes in a cell at the transcriptional, co-transcriptional and posttranscriptional levels. Among all classes of short ncRNA identified to date, miRNAs role in cancer has been investigated most thoroughly .Single or clustered genes encoding primary miRNA transcripts (pri-miRNAs) ultimately processed into short, ~22 nucleotide sequences, are dispersed throughout the genome and mostly conserved among species. Transcribed by RNA Polymerase II, pri-miRNAs undergo processing by Drosha complex in the nucleus. The processed long miRNA precursors (pre-miRNAs) are exported to the cytoplasm by exportin 5 and cleaved into double-stranded short precursors of miRNAs. After a double-stranded miRNA is loaded into RISC complex, one of the RNA strands, the passenger, is removed, allowing the seed sequence of miRNA to pair with mRNA targets. The main characteristic of miRNA gene silencing pathways is that the single-stranded miRNAs facilitate translational repression and mRNA destabilization through imperfect base-pairing typically with the 3′UTRs.miR-15a-1 and miR-16b-1 was missing in 70% of B-cell chronic lymphoblastic leukemia with translocation at 13q14. Several powerful oncogenes promoting CLL such as Cyclin D1, MCL1, and anti-apoptotic factor BCL2, were identified as the downstream targets of miR-15a-1 and miR-16b-1 [The first evidence of miRNA gene dysfunction provoking a blood malignancy was reported in 2002 by Calin et al. . The poliR-16b-1 .miR-145 and miR-146a, which are dysregulated in 5-q syndrome, a subtype of MDS characterized by severe anemia, variable neutropenia, and atypical megakaryocytes. The deletion of 1.5 Mb DNA on the long arm of chromosome 5 (del5q) leads to miR-145 and miR-146a loss-of-function and a subsequent upregulation of Toll–interleukin-1 receptor domain–containing adaptor protein (TIRAP) and tumor necrosis factor receptor–associated factor-6 (TRAF6), triggering phenotypical and functional features of MDS [Another vivid example of tumor suppressor miRNAs are s of MDS .C13orf25  encoding the miR-17-92 cluster was found in diffuse large B-cell lymphoma, the cancer of immune cells residing in the lymph nodes. Also known as oncomiR-1, the well-studied miR-17-92 cluster consists of six miRNAs  important for cell cycle, proliferation, apoptosis, and other pivotal processes. The miR-17-92 cluster is often dysregulated in hematopoietic and solid cancers. Transactivated by c-MYC, N-NYC, MXI1, and other transcription factors, miR-17-92 cluster increases cell proliferation and survival by inhibiting several critical tumor suppressors such as PTEN and pro-apoptotic factor p21 [The amplification of genomic loci ctor p21 .miR-29 . However, miR-29s role in blood and other malignancies is dual as they can act as oncogenes or tumor-suppressors in different histological types of tumors [miR-125b overexpression is shown to induce either myeloid or lymphoid leukemia depending on the time course and expression levels of miR-125b [miR-155 to high levels (>50-fold above controls) displayed antitumor activity in different types of AML . Conversely, moderate upregulation of miR-155 was associated with alternative target selection, repression of myeloid differentiation genes, and with leukemic phenotypes in vitro and in vivo [MiR-126 regulates quiescence and self-renewal in normal and malignant human hematopoietic stem cells with distinct outcomes [miR-126 promote leukemogenesis in AE9a-induced mouse model [Another miRNA family playing an essential role in AML, CLL, and lymphomas is f tumors . The conf tumors ,161. FormiR-125b ,163. Nar in vivo . MiR-126outcomes ,166. Surse model .Cyclin D1 and unfavorable prognosis [Aberrant expression of miRNA in various subtypes of myeloid and lymphoid leukemia was extensively investigated, and thoroughly reviewed ,170,171.rognosis . By examrognosis . Since mrognosis , indicatSimilar to other classes of ncRNA, abnormal miRNA expression and processing in cancer are caused by structural and functional changes in the human genome: chromosomal rearrangements, deletions, amplifications, and deregulated epigenetic and transcriptional control of gene expression. Although copy number alterations (CNAs), amplification, and deletions are powerful genetic mechanisms of miRNA deregulation, they are not quite common for AML. By studying 113 cases of AML, Ramsingh et al. show that only 18% of patients have CNAs involving miRNA genes, while multiple alterations in epigenetic and transcriptional regulators are in charge of miRNA abnormal expression .miR-16-1–miR-15a that alter the processing of these miRNAs and can cause loss of function similar to a deletion [Germline variants in miRNA genes may have a profound effect on miRNA transcription and maturation ,177; howdeletion . Accordideletion ,183.Traditionally, therapeutic approaches for targeting a primary RNA structures were based on introduction of complementary DNA or RNA oligonucleotides, or their chemical equivalents, into the target cells. Oligonucleotides can function through RNase H-mediated RNA degradation, RNA interference (RNAi), or through a non-degradative steric hindrance mechanism by replacing or repressing RNA-binding proteins .miR-126 targeting by the transferrin or anti-CD45.2 antibody-conjugated nanoparticles containing antagomiR-126 both in vitro, in CD34+ blasts sorted from primary elderly AML patients, and in vivo, using Mll PTD Flt3 ITD mouse model [miR-34a, which directly regulates at least 24 known oncogenes. At some point, the trial was stopped due to life-threatening immune responses in several patients, but, ultimately, the study was competed using dexamethasone premedication and dose-escalation protocols. Overall, MRX34 demonstrated an acceptable safety for most of the patients and showed the evidence of antitumor activity in a subset of patients with refractory tumors [Meant to silence gene expression by inducing degradation of target mRNAs, double-stranded siRNAs and single-stranded antisense oligonucleotides (ASOs/AONss) are designed to perfectly match the target sequence. Synthetic miRNAs are introduced into a cell either to replace downregulated endogenous miRNAs (RNA mimics) or block the endogenous miRNAs, which resembles an antisense approach. Dorrance et al. demonstrated a successful se model . Antagomse model . While mse model . The firse model . MRX34 wy tumors .BCR-ABL. While some studies reported a significant improvement in survival for particular groups of patients [lnc-THADA4-1 in Juvenile Myelomonocytic Leukemia (JMML) [BCR-ABL in CML [The 18 clinical trials of anti-sense therapies in chronic and acute leukemias targeted exclusively transcriptional regulators, mostly patients , AONs stpatients . The proa (JMML) , and antL in CML , suggestDelivery efficiency remains one of the important problems in nucleotide-based therapies. Therapeutic molecules can be trapped in endosomes, lysosome or disposed through exocytosis and, therefore, remain inactive ,192. DelSeveral commercially viable AON-based therapies are currently FDA approved, and are aimed to treat cytomegalovirus (CMV) retinitis, common in people with a compromised immune system, and hereditary conditions such as Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) . In the A combined high-throughput screening of antisense oligonucleotides and small molecules identified compounds promoting exon 51 skipping in dystrophin pre-mRNA . Similarpre-miR-21 and inhibit miR-21 biogenesis. In vitro, these compounds, e.g., mitoxantrone, reduced mature miR-21 levels and modulated miR-21-mediated invasive phenotype. Importantly, the chemical crosslinking and a pull-down assay (Chem-CLIP) studies confirmed physical interaction between pre-miR-21 and the small molecule. Among different classes of compounds, topoisomerase inhibitors, kinase inhibitors, and splicing modulators were key classes that bound RNA [Velagapudi et al. investigated oncogenic non-coding RNA targeting by known anti-cancer drugs . The teaound RNA .The high-throughput methods for investigating chemical compounds targeting RNA molecules and mechanisms of drugs targeting RNA–protein interactions were recently reviewed. Anita Donlic and Amanda Hargrove placed a unique emphasis on the specifics of RNA structural elements or RNA-mediated interactions that enable disease-related functions in mammalian systems as well as the phenotypic changes observed upon treatment with targeted ligands . Zhu et Once defined as architects of eukaryotic complexity and the dark matter of cancer genomes ,205, ncRAnother level of RNA network complexity lays in the abundance and variety of ncRNA interactions with mRNA and other ncRNA molecules. The multifaceted ncRNAs acting as transcriptional, co-, and posttranscriptional regulators indicate the importance of understanding the circuitous architecture of the RNA network. Although the selective targeting of upregulated oncogenic RNA molecules may seem a step towards personalized medicine, in most clinical settings only a limited number of patients respond to targeted therapies that address a single genetic abnormality . Thus, tcis- and trans-acting RNA regulatory elements and RBPs in human disease might be larger than currently known.Understanding the functional significance of the somatic point mutations and genomic variants located in non-coding and untranslated regions of the genome is also a challenge since they can influence the expression of distal genes at both transcriptional and posttranscriptional levels. Annotation of twenty-three million regulatory SNPs that are involved in a wide range of processes, including proximal and distal transcriptional and posttranscriptional regulation of gene expression, indicates that roughly half of them are involved in RBP- and miRNA-mediated posttranscriptional regulation . A globaThe concept of RNA-targeting therapeutics using ASO, siRNA, miRNA and other synthetic RNA has been proven to be effective in some degenerative diseases. The efficient and safe targeted delivery of RNA therapeutics into specific tissues will be key for expanding those approaches to other clinical indications including cancer. Recent discoveries in the chemical targeting of RNA motifs and identification of small molecules disrupting RNA–protein and RNA–RNA interactions open a new area in RNA therapeutics that may help in developing a next generation of anti-cancer drugs.


A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types.Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3′ splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs.This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing. RNA can act as a carrier of information from the nucleus to the cytoplasm in the processing of protein-coding genes, as a regulatory molecule that can control gene expression, and even as an extracellular signal to coordinate trans-generational inheritance –3. RNA bTo identify direct RNA targets of RBPs, RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP) methods are frequently used. CLIP-based methods utilize UV crosslinking to covalently link an RBP with its bound RNA in live cells, enabling both stringent immunoprecipitation washes and denaturing SDS-PAGE protein gel electrophoresis and nitrocellulose membrane transfer which serves to remove background unbound RNA . AnalyseThe field of transcription regulation observed similar challenges and opportunities in integrating transcription factor target profiles . To addrTo this end, we introduced the enhanced CLIP (eCLIP) methodology featuring a size-matched input control  and charIn this companion work, we provide further insight into how integrative analysis of RBP target profiles by eCLIP can reveal both general principles of RNA processing as well as specific mechanistic insights for individual RBPs. Although most CLIP analysis typically focuses on binding to mRNAs (both intronic and exonic), we find that for 70% of RBPs, the dominant enrichment signature is instead a variety of multicopy and non-coding elements . These analyses can be then used to generate hypotheses about RBP function, as enrichment for the ribosomal RNA precursor corresponds with RBPs regulating ribosomal RNA maturation whereas enrichment for retrotransposable elements corresponds to both regulation of retrotransposition itself as well as suppression of improper RNA processing due to cryptic elements contained within these elements. Binding maps across meta-profiles of mRNAs and exon-intron junctions similarly show that RBP binding patterns correlate with RBP functional roles, and analysis of spliceosomal components indicates that eCLIP can be used to identify branch points and provides evidence for a 3′ splice site scanning model. In summary, these results provide further validation of the power of integrated analyses of RBP target maps generated by eCLIP in identifying novel principles of RNA biology, as well as generating RBP-specific hypothesis for further functional validation.https://www.encodeproject.org) [The eCLIP methodology enabled highly efficient identification of RBP binding sites , leadingect.org) .Fig. 1T32P to visualize protein-RNA complexes after SDS-PAGE electrophoresis and membrane transfer in order to query whether RNA bound to co-purified RBPs of different size is present [32P while using commercially available chemiluminescent detection reagents for biotin-labeled nucleic acids , this labeling uses the standard eCLIP RNA adapter ligation reaction and thus may more accurately reflect true protein-coupled RNA positioning.Many CLIP methods included radioactive labeling of the 5′ end of RNA fragments with  present . HoweverSurprisingly, when expanding this approach across RBPs, we observed detectable transfer of RNA from non-crosslinked cells to nitrocellulose membranes in a supplier-dependent manner  among all peak-containing genes . In particular, well-studied splicing regulators  were more than 3-fold enriched for binding to RBPs , 7SK snRNA, and others that have one or few expressed primary transcripts but dozens to hundreds of pseudogenes throughout the genome, as well as retrotransposable elements including LINE and Alu elements with thousands of moderately divergent sense and antisense copies throughout transcribed genes . We founIn order to include these RNA types in eCLIP analysis, we developed a “family-aware mapping” approach in which adapter-trimmed reads are first mapped against a database of sequences for primary transcripts and pseudogenes for 82 families Fig. . Reads mAt the element level, our family-aware mapping strategy recovers many known processing or interacting factors, including RBPs enriched for the mature 18S RPS3, RPS11) and 28S rRNA  as well as the 45S rRNA precursor , tRNAs (NSUN2), RN7SK (LARP7), YRNA (TROVE2), and others  is the most abundant RNA found in eukaryotic cells and plays essential roles in defining the structure and activity of the ribosome. In humans, the 5S rRNA is separately transcribed, whereas the 18S, 28S, and 5.8S rRNAs are transcribed as one 45S precursor transcript that then undergoes a complex series of cleavage and RNA modification steps to process the mature rRNAs, which then form complex structures that scaffold the assembly of ~ 80 proteins to create the functional ribosome . Unbiasep = 0.00025 by Fisher’s exact test)   or the 45S precursor (R2 = 0.17)  Fig. h, providRepetitive elements constitute a large fraction of the non-coding genome , and eleQuerying for RBPs with enriched eCLIP signal at retrotransposable and other repetitive elements, we surprisingly observed that only a small subset of elements (notably including L1 and Alu elements both in sense and antisense orientation) showed high RBP specificity, whereas most elements showed extremely highly correlated enrichments across RBPs , PPIL4 (0.06), and TRA2A (p = 0.05) were all identified as candidate suppressors of L1 retrotransposition in a recent genome-wide CRISPR screening assay [Considering L1/LINE elements, we observed enrichment with far more RBPs, with 26 datasets showing 5-fold enrichment Fig. c. Intereng assay , suggestHowever, we observed that while enriched signal was centered at L1 sense and antisense elements, the signal often extended for multiple kilobases on either side  density or biased towards the 5′ (CDS.5P) or 3′ (CDS.3P) end. We observed that 13 out of 15 spliceosomal RBPs showed CDS enrichment (10 of which fell into the CDS.UN category), likely reflecting the general lack of introns in 5′UTRs  and 3′UTRs (as they would create targets for nonsense-mediated decay)  the stop codon, including the well-studied IGF2BP family of RBPs  and 500 nt of proximal introns flanking both the 3′ and 5′ splice sites. As an example, we observed that out of 89,265 introns present in highly expressed transcripts (TPM > 1), 2699 had a significant IDR peak from eCLIP of U2AF2 in K562 cells . The overall snRNA family includes five specific RNA families  that play essential roles in canonical GT-AG RNA splicing, as well as four  specific to the minor AT-AC spliceosome, each of which plays specific mechanistic roles during splicing . Thus, RInterestingly, while many factors showed similar association between analogous snRNAs in the major and minor spliceosomes , some RBPs were specifically associated with either the major  or minor spliceosome   studies focusing on one predicted novel interaction group involving HNRNPL and AGGF1. We observed that AGGF1 co-immunoprecipitated HNRNPL, unlike unrelated factors RBFOX2 or FMR1 . We also observe classes of RBPs with broader patterns of enrichment, with a particularly interesting group showing a stereotypical pattern of high enrichment at the 5′ end of introns (extending hundreds of bases into the intron). Notably, this cluster contains multiple factors with links to co-transcriptional RNA processing, including CSTF2T , XRN2 6, and NonThe enrichment for previously identified rRNA processing factors suggests that many additional factors here may represent unexplored regulators. Indeed, building upon rRNA enrichments observed from the analyses described here, further research has led to validation of NOL12  and AATFAnother benefit of the unbiased approach presented here is that it enables identification of novel potential sites of regulatory activity, as our analysis of the 45S ribosomal RNA precursor indicates a surprising cluster of substantial RBP eCLIP enrichment at an uncharacterized region located between the A0 and 1 processing sites. This region (particularly the sharp peak observed in LIN28B eCLIP) is centered on a putative ribosomal-encoded microRNA rmiR-663) , and our , and ouAnalysis of Alu elements identified 3 RBPs with at least 4-fold enrichment, each of which appears to reflect a different underlying mechanism. The most enriched RBP, HNRNPC, has previously been shown to suppress cryptic 3′ splice site signals in antisense Alu elements . In contSimilarly, analysis of L1 element enrichment revealed multiple modalities of regulatory activity. One function of RBP association to L1 is to suppress retrotransposition activity, and indeed, we observed that three RBPs  showed both eCLIP enrichment for sense L1 elements and act to suppress L1 retrotransposition activity in genome-wide screening data. For RBPs enriched for antisense L1 elements, we instead see signatures of RBPs acting to increase RNA expression, extending a similar analysis recently published  that revealed widespread association with L1 elements by RBPs . From thIt is notable that the above enriched RNA element classes often reflected a substantial fraction of eCLIP reads, suggesting that they may represent dominant functions of the RBP. For example, antisense L1 elements constituted 19–27% of eCLIP reads for HNRNPM and MATR3 and antisense Alu elements were 13–18% of reads in HNRNPC eCLIP. Similarly, 42–56% of UTP18, 27–31% of WDR43, and 16% of HepG2 LIN28B eCLIP reads mapped to the 45S ribosomal RNA precursor. Thus, these results strongly argue that analysis of CLIP data should include proper quantitative analysis of reads mapping to non-mRNA regions, as they can in some cases represent the dominant binding modality of the RBP and should be considered in interpreting potential functional roles of the RBP in regulating RNA processing.Intriguingly, we even observed significant differences even between RBP components of the same RNP complex. For example, 41.0% of SF3A3 HepG2 eCLIP reads mapped to RNU2 snRNA versus only 8.5% mapping to proximal intronic regions; in contrast, SF3B4 was far more even . Although we cannot rule out that this difference in crosslinking to snRNA versus intron reflects underlying amino acid biases in UV crosslinking efficiency, it does confirm that CLIP profiling of multiple RBP members of an RNP complex can yield distinct insights into interaction patterns and regulatory roles of the complex, suggesting that it is critical to assay multiple independent proteins to gain a full understanding of the target repertoire of an RNP complex.In addition to specific insights into the RBPs themselves, we anticipate that the broad diversity of RBPs profiled and RNA elements and features bound will spur further development of methods targeted towards specific RNA processing steps. For example, the peak distribution pattern of the CDS.5P class (and RPS3 in particular) resembles the average profile observed using ribosome profiling , suggestXist and Malat1 also suggested that the patterns of RBP enrichment often correlate with specific structural and functional domains on these non-coding RNAs [The diversity of distinct RBP association patterns can also be flipped to predict features of a queried RNA. For example, recent work used the ENCODE eCLIP resource to identify UPF1 as one of many RBPs with specific enrichment at 3′UTRs . This fiing RNAs . With thThe maturation of methods to profile the in vivo targets of regulatory proteins at both the DNA and RNA level has enabled unparalleled large-scale efforts to map the human gene expression regulatory network , 20. In https://www.encodeproject.org) with accession identifiers listed in Additional file p value ≤10−3) in immunoprecipitation versus paired size-matched input. RNA binding protein function annotations and localizations were obtained from [Enhanced CLIP (eCLIP) datasets used were obtained from the ENCODE data coordination center , centrifuged at 15k RPM for 3 min at 4 °C (with supernatant kept) to clear lysate, and incubated with rotation overnight with antibody coupled to species-specific secondary beads . After incubation, samples were washed once with eCLIP wash buffer, washed twice with high-salt wash buffer, and washed three times with wash buffer. FastAP and T4PNK reactions were performed on-bead as previously described for eCLIP, followed by one wash with high-salt wash buffer and 3 washes with wash buffer. At this point, a modified RNA linker ligation was performed with standard eCLIP ligation conditions (buffer and High ?A3B2 show $132#?>Concentration T4 RNA Ligase) but with 500 pmol pCp-Biotin (Jena Bioscience) in place of the RNA adapter, and samples were incubated at 16 °C. For some experiments, immunoprecipitations were performed on 4 million cells; for these experiments, half reactions were used for the pCp-biotin ligation step. After ligation, samples were washed once with high-salt wash buffer and three times with wash buffer, followed by standard SDS-PAGE electrophoresis and transfer to nitrocellulose membranes. Visualization was performed using the Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher), following the manufacturer’s instructions for blocking, washes, and labeling. Imaging was performed on the Azure C600 platform. For 32P experiments, radiolabeling was performed as previously described [A step-by-step version of the biotin-based labeling protocol is available at escribed .https://github.com/YeoLab/repetitive-element-mapping, and was initially described in [The software pipeline used to quantify enrichment for retrotransposable and other multicopy elements is available at ribed in  but is dk-mers, and 49 families comprising 705 total human repetitive elements obtained from the RepBase database (v. 18.05) [A database of multicopy elements was generated based on 5606 transcripts obtained from GENCODE v19 covering 34 families of abundant non-coding, multicopy, and other types of RNA refractory to standard peak analysis, including families within the broader rRNA , snoRNA , snRNA , vault RNA , non-coding RNA , and small Cajal body-specific RNA (SCARNA) broader classes  . Within Q, 40.0)/40.0)) where Q is the Phred quality value, and default values MX = 6, MN = 2, as described in bowtie2 reference material) and gap penalties  were kept. Next, the mapping to the transcript with the highest priority within a RNA family (as listed above) was identified as the “primary” match mapping. At this stage, read pairs which had equal best alignments to multiple repeat families were discarded, with only reads mapping to a single repeat family considered for further quantification.To quantify eCLIP signal, paired-end sequencing reads were first adapter trimmed as previously described . Next, rNext, these RNA family mappings were integrated with unique genomic mapping from the standard eCLIP processing pipeline . For read pairs that mapped both to an RNA family above as well as uniquely to the genome, the mapping scores (as defined above) were compared. If the unique genome mapping was more than 2 mismatches per read  better than to the repeat element, the unique genomic mapping was used; otherwise, it was discarded and only the repeat mapping was kept. Next, PCR duplicates were removed by comparing all read pairs based on their mapping start and stop position (either within the genome or within the mapped primary repeat) and unique molecular identifier sequence, and all but one read pair for read pairs sharing these three values were defined as PCR duplicates and removed. At this stage, RepeatMasker-predicted repetitive elements in the hg19 genome were additionally obtained from the UCSC Genome Browser [pi and qi are the fraction of total reads in IP and input respectively that map to element i. To combine two biological replicates, the average reads per million (RPM) was calculated across two IP samples and compared against the paired input experiment to calculate one overall fold-enrichment and relative information value per dataset.Finally, the number of post-PCR duplicate removal read pairs mapping to each class was counted in both IP and paired input sample and normalized for sequencing depth . Significance was determined by Fisher’s exact test or Pearson’s chi-square test if all expected and observed values were five or more. Relative information content of each element in each replicate was calculated as To quantify whether RNA element enrichment matched with RBP functions, a set of positive control pairings were generated between RNA elements with known links to either RBP function or known RBPs contained within a well-characterized ribonucleoprotein complex  were obtained from the UCSC Genome Browser, along with 100 nt of flanking sequence. Only reads that perfectly aligned (with zero mismatches or gaps) to these sequences were counted for further analysis.p values using n = 15 (the number of L1 (sense)-enriched RBPs queried).L1 retrotransposition genome-wide CRISPR screening data was obtained from Liu et al. , using Chttp://www.encodeproject.org) for all RBPs with both eCLIP and RNA-seq performed in the same cell type. L1 sense and anti-sense elements were taken from RepeatMasker-predicted repetitive elements in the hg19 genome obtained from the UCSC Genome Browser [To calculate change in expression of L1-containing bound genes, DESeq-calculated gene expression fold changes for RBP knockdown/RNA-seq data were obtained from the ENCODE DCC  to sequences present in RepBase, whereas “divergent” reads mapped better to unique genomic loci than to the reference sequence.Calculation of overall element coverage  and K562  was chosen as the representative transcript, and the set of expressed genes  were considered. Datasets with fewer than 100 mRNA-overlapping peaks were discarded, leaving 205 datasets. Next, each gene was split into 162 bins , based on the median 5′UTR, CDS, and 3′UTR lengths of highly expressed (TPM ≥ 10) GENCODE v19 transcripts in K562 cells. For each eCLIP dataset, the average peak coverage for each bin was calculated for each gene and then averaged over all genes to generate final meta-gene plot. To generate confidence intervals, bootstrapping was performed by randomly selecting (with replacement) the same number of transcripts and calculating the average position-level peak coverage as above, with the 5th and 95th percentiles (out of 100 permutations) shown. For further visualization and analysis, only 104 RBPs where the 5th percentile was at least 0.002 peaks per gene (~ 20 peaks in at least one bin) were considered. Normalized coverage was then calculated by setting the maximum position to one and minimum position to zero for each eCLIP dataset. Cross-position correlations were calculated using normalized coverage for across all 104 RBPs at each position. Odds ratios and significance  utilized RBP annotations , the region from 500 nt upstream to 500 nt downstream  was queried for the presence of significant (IDR) peaks. Finally, the number of peaks at each position was averaged over all events to obtain the final meta-exon value. To generate confidence intervals, bootstrapping was performed by randomly selecting (with replacement) the same number of transcripts and calculating the average position-level peak coverage as above, with the 5th and 95th percentiles (out of 100 permutations) shown. For further analysis, only datasets with at least 100 IDR peaks were considered. Next, after calculating meta-exon profiles and confidence intervals as above, datasets that did not have at least one position with the 5th percentile bootstrap value above a minimal cutoff of 0.0005 (~ 5 peaks observed at that position) were discarded to leave 133 datasets for further consideration. Finally, for visualization of comparison across RBPs Fig. , an addiTo identify points of enriched read termination in AQR eCLIP, regions from − 50 nt to − 15 nt from annotated 3′ splice sites were obtained from GENCODE v19, and the subset of regions with at least 20 overlapping reads in AQR eCLIP in K562 cells were taken for further analysis. Points of enrichment were identified as those where more than half of reads overlapping the overall region terminated at the same position. Motif analysis was performed by counting the frequency of 11-mers centered on the read start position with 5 nt flanking on either side. Motif logos were generated with seqLogo (R).Splicing maps profiling normalized enrichment for SF3B4 and SF3A3 at RBP knockdown-responsive alternative 3′ splice site events were generated as previously described , 76. In Overlap between eCLIP datasets A and B was determined by calculating the fraction of significant and reproducible peaks in dataset A that overlapped (by at least one base) a peak in dataset B, and vice versa the fraction of peaks in B that overlapped a peak in A, and taking the maximum of those fractions as the overall pairwise fraction overlap. Only datasets with at least 100 reproducible and significant peaks were used for this analysis. Gene Set Enrichment Analysis was performed using the GSEA software package . RBP intIP-western validation was performed using HNNRPL , RBFOX2 , FMR1 , AGGF1 , and TNRC6A  antibodies in UV crosslinked K562 cells. Immunoprecipitation in high-salt wash conditions was performed using standard eCLIP wash buffers, beads, and other reagents . Low-salAdditional file 1: Table S1. Accession identifiers for eCLIP datasets used in the manuscript.Additional file 2: Table S2. RNA binding protein function annotations, localization patterns, and predicted RNA binding domains.Additional file 3: Supplementary Figures S1-S8.Additional file 4: Table S3. List of multi-copy element annotations used in family-aware mapping.Additional file 5: Table S4. Quantitation of multi-copy RNA family enrichment for 223 eCLIP datasets.Additional file 6: Review history.


RNA sequencing (RNA-seq) has matured into a reliable and low-cost assay for transcriptome profiling and has been deployed across a range of systems. The computational tool space for the analysis of RNA-seq data has kept pace with advances in sequencing. https://doi.org/10.1128/mSystems.00917-20), demonstrates that integrating prokaryote-specific strategies into existing RNA-seq analyses improves read quantification. Unlike in eukaryotes, polycistronic transcripts derived from operons lead to sequencing reads that span multiple neighboring genes. Chung et al. introduce FADU, a software tool that performs a correction for such reads and thereby improves read quantification and biological interpretation of prokaryotic RNA sequencing.RNA sequencing (RNA-seq) has matured into a reliable and low-cost assay for transcriptome profiling and has been deployed across a range of systems. The computational tool space for the analysis of RNA-seq data has kept pace with advances in sequencing. Yet tool development has largely centered around the human transcriptome. While eukaryotic and prokaryotic transcriptomes are similar, key differences in transcribed units limit the transfer of wet-lab and computational tools between the two domains. The article by M. Chung, R. S. Adkins, J. S. A. Mattick, K. R. Bradwell, et al. (mSystems 6:e00917-20, 2021,  Over the last 15 years, RNA sequencing (RNA-seq) has offered a high-resolution view of the presence and abundance of transcripts at a given time . Transcr4–Fundamental differences in transcription limit the transfer of innovation in the quantification of transcripts between biological domains. Eukaryotic transcripts contain a single product (monocistronic), while many prokaryotic transcripts contain multiple products . Computational prediction of operon structures is still an active area of research, meaning that laboratory-based techniques like 5′ and 3′ rapid amplification of cDNA ends (RACE) and direct RNA sequencing remain the gold standard for operon prediction but preclude application to the majority of RNA sequencing experiments. Without well-annotated reference transcriptomes that contain polycistronic transcripts, genome-based alignment strategies better capture reads that span genes in an operon. Similar to multimapped reads, reads that span multiple genes create problems in read quantification. However, as a uniquely prokaryotic problem, this problem has received little attention.With the development of the FADU software tool, Chung and colleagues  present This correction is a valuable contribution that is poised for incorporation into general prokaryotic RNA-seq analyses as well as the broader read counting tool space. As a stand-alone tool that operates on BAM alignment files, FADU can be integrated into RNA-seq analysis as an alternative to software like featureCounts or HTSeq. FADU has a small memory and CPU footprint that is permissive for integration into routine RNA-seq analysis pipelines, including for large-scale RNA-seq analyses. Alternatively, many other tools that perform read quantification already provide optional parameters to control behavior around multimapped reads and thus likely contain the infrastructure to support the adoption of this new correction technique , 10. IntWhile Chung and colleagues  explored


While the current COVID-19 pandemic continues, the US Food and Drug Administration (FDA) has approved only one drug against the virus—remdesivir. It is a nucleotide analogue inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase; favipiravir is another member of the same class. These nucleoside analogs were originally developed against other viral polymerases, and can be quickly repurposed against SARS-CoV-2 should they prove efficacious. We used cryoEM to visualize how favipiravir-RTP binds to the replicating SARS-CoV-2 polymerase and determine how it slows RNA replication. This structure explains the mechanism of action, and will help guide the design of more potent drugs targeting SARS-CoV-2. The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses. The COVID-19 coronavirus pandemic has spurred research into novel and existing antiviral treatments. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA-dependent RNA polymerase (RdRp), crucial for the replication and transcription of this positive-sense single-stranded RNA virus, is a promising drug target for the treatment of COVID-19 –5. RecenFavipiravir (T-705-RTP) was purchased from Santa Cruz; upon receipt, a stock solution of 50 mM T-705-RTP resuspended in 100 mM Tris, pH 7.5 buffer was aliquoted and stored at 31P-NMR spectra of the T-705-RTP stock solution were acquired using a Bruker Ultrashield 400 Plus operating at 162 MHz.Mass spectra were acquired on an Agilent 1200 liquid chromatography–mass spectrometry system equipped with an electrospray ionization source and a 6130 quadrupole spectrometer  encoding a SARS-CoV-2 RdRp polyprotein, codon optimized for pression . The celEscherichia coli BL21(DE3) Star cells cotransformed with the nsp7 and nsp8 plasmids. Cells were grown in ZY autoinduction media , and a pET28 vector modified to encode an N-terminal 6×-His-Sumo tag in frame with the multiple cloning site. Protein expression was pursued in on media  at 37 °Cm/z . RNAs were cleaved from the solid support by treating with 4 mL of a 1:1 mixture of 28% wt Single-stranded primer and template RNAs A were rePurified apo-RdRp, assembled as above, was mixed immediately after purification with annealed dsRNA, resuspended in SI Appendix, Fig. S1) were performed at room temperature in assembly buffer. Reaction conditions were designed to approximate, as closely as possible, those employed in production of vitrified electron microscopy (EM) grids. For primer extension assays, 6 All assays  were run at 15 W in Tris–borate–EDTA buffer for 2 h. The RNA gel was stained using SYBR Gold Nucleic Acid Gel Stain (Invitrogen). Fluorescence imaging was performed using an Amersham Typhoon imager (Cytiva).The complex, prepared as described above, was used at a concentration of 1.3 mg/mL for preparing cryoEM grids. All-gold HexAuFoil grids with a hexagonal array of 280-nm-diameter holes, 700-nm hole-to-hole spacing, and 330 Å foil thickness were made in-house, and plasma was treated as described in ref. cryostat , and werSI Appendix, Table S1). Other surfaces that were screened during initial specimen preparation included graphene functionalized with amylamine or hexanoic acid and graphene oxide, none of which improved the orientation distribution or yielded two-dimensional (2D) classes indicating degradation of the complex more severe than due to the air–water interface alone. Thus, they were not included in the dataset for high-resolution reconstruction. The high density of foil holes on the HexAuFoil grid, combined with fast imaging by aberration-free image shift and a fast, direct-electron detector , allowed us to acquire more than 200 micrographs per hour for 14 consecutive days. The number of micrographs that can be acquired from a single HexAuFoil grid is limited only by the ice contamination rate in the vacuum of the electron microscope, rather than by the number of holes on one grid, even with the fastest available data collection setups.We acquired electron micrographs from four grids: three HexAuFoil grids  and one partially hydrogenated graphene-coated grid   with manual retraining of the model. After four rounds of 2D classification, it was clear that the specimen adopted a preferred orientation . E. EEod, isurfaces . The atosurfaces , with masurfaces , 26, andsurfaces .SI Appendix, Fig. S1) . The formation of larger RNA products observed at later time points  must arise through promiscuous base pairing with both uracil and cytosine in the template strand, and is consistent with the reported mutagenicity of favipiravir toward viral genomic RNA in vivo . Finally, we tested whether preincubation with favipiravir-RTP inhibited primer extension in the presence of rNTPs. Despite the low incorporation of favipiravir-RTP observed in primer extension assays, it nevertheless suppressed completion of RNA replication (as judged by the absence of the full 31mer primer extension product) at all concentrations tested, even when rNTPs were added at a considerable excess over the inhibitor . The accumulation of shorter primer extension intermediates at higher concentrations of favipiravir-RTP may indicate premature replication termination. These assays are thus consistent with a previously proposed mode of action of favipiravir-RTP, comprising, in addition to mutagenesis, both slowed RNA synthesis and nonobligate chain termination  . The overall structure of the polymerase complex observed , comprising one nsp12, one nsp7, two nsp8 subunits, and a template:primer RNA, is nearly identical to those previously described . Favipiravir is stacked onto the 3′ nucleotide of the primer strand, and forms a noncanonical base pair, through its amide group, with the displayed cytosine in the template RNA strand, consistent with favipiravir mimicking a guanosine base. The weaker density for the inhibitor, compared to that for the neighboring RNA bases, indicates partial occupancy of the site. The density is most consistent with the inhibitor binding noncovalently to the polymerase, with little to no covalent incorporation. The biochemical assays indicated only minimal incorporation of favipiravir-RTP into the primer under the conditions used to prepare the complex for structural analysis . We also observed a weak signal, adjacent to the trisphosphate moiety of favipiravir-RTP, consistent with a pyrophosphate molecule . We show that pyrophosphate does not inhibit RdRp activity in the presence of rNTPs . Pyrophosphate was modeled at this site in the SARS-CoV-2 RdRp nsp12–RNA–remdesivir complex (PDB ID code 7BV2)  . Howeverde 7BV2) ]. In conWe observe signal for the RNA base pairs as synthesized, indicating no translocation of the RNA has occurred in the majority of the intact complexes during the incubation period of 30 min. This supports the interpretation that favipiravir stalls replication by noncovalent interactions at the active site, rather than by covalent incorporation into the replicating strand. Neither base stacking nor base pairing distances in the presence of favipiravir differ from those expected for natural nucleotides. In addition to base pairing with cytosine from the template strand, favipiravir is coordinated by Lys545 in the F1 domain of nsp12, which is positioned to accept hydrogen bonds from the nitrogen atom in the pyrazine ring or donate to the fluorine atom of the inhibitor, although, in both instances, the distances are long , suggesting possible water-mediated contacts A. These SI Appendix, Fig. S4). A similar conformation of favipiravir-RTP   showed tavir-RTP C would aon assay . We theron assay C.SI Appendix, Fig. S7) indicate that pyrophosphate at concentrations between 10 and 500 One might hypothesize that pyrophosphate contributes to the slow rate of favipiravir-RTP incorporation by directing the triphosphate moiety of favipiravir-RTP into a nonproductive binding mode. However, pyrophosphate inhibition assays  . This maMultiple factors, including complex dissociation, denaturation at the air–water interface, and preferred orientation, impeded our initial attempts for high-resolution structure determination of this complex. We managed to circumvent these by acquiring a large cryoEM dataset and using a combination of HexAuFoil and partially hydrogenated graphene-coated UltrAuFoil EM grids. We make the complete dataset publicly available via the Electron Microscopy Public Image Archive (accession number 10517) in the hope that it will be useful for software development for handling large datasets of structurally heterogeneous cryoEM specimens. For now, cryoEM structure determination of this RdRp–RNA complex in combination with different inhibitors remains far from a routine task. Still, this and other published structures of the complex will aid rational drug design. In the interim, biochemical optimization of the stability of the complex might help yield a specimen that is more stable and randomly oriented in the thin layers of buffer used for cryoEM. This would then make it suitable for rapid structure determination among an array of candidate compounds. The need for a derivatized graphene surface to overcome the strong interactions with the air–water interfaces highlights the problem that surfaces present in cryoEM specimen preparation; further improvements in the specimen preparation process are still needed to enable the high throughput required for drug design and analysis.The structure reported here suggests at least three possible directions for further efforts toward drug design against the SARS-CoV-2 RdRp. The first is modification of the favipiravir-RTP to bring the l genome . Second,Supplementary File


We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to  Complete loss of SE expression as in the null mutant se-4 is embryonic lethal, while the se-1 mutation, which results in the expression of a truncated SE lacking 20 amino acids at its C-terminus, severely affects developmental timing, phylotaxy, meristem function and patterning of leaves and flowers  tail at the 3′ end , and som flowers . The se- ago1-25 .In addition to its role in miRNA biogenesis, Arabidopsis SE contributes to both constitutive and alternative splicing of pre-mRNAs ,15, enhaSchizosaccharomyces pombe, homologs of MTR4, ZFC3H1 and PAPBN1 form a similar complex named MTREC (Mtl1-Red1-Core) or NURS (nuclear RNA silencing) which is required for the elimination of meiotic mRNAs during mitosis and the degradation of unspliced mRNAs, non-coding RNAs and cryptic intergenic transcripts (PAXT connects the RNA helicase hMTR4 and the Zn-knuckle protein ZFC3H1 with the nuclear poly(A)-binding protein PABPN1, and promotes the degradation of transcripts with long poly(A) tails in the nucleoplasm. In nscripts ,30. The nscripts . Both PAnscripts . The curnscripts ,34.In a previous study, we demonstrated that the Arabidopsis RNA exosome complex is associated with two nuclear RNA helicases, AtMTR4 and HEN2 . AtMTR4 in vivo, and investigate the individual interactions between SE and subunits of NEXT. We show by yeast two hybrid (Y2H) assays, FRET-FLIM and reciprocal co-immunoprecipitation (co-IP) experiments that the two ZCCHC8A and ZCCHC8B subunits directly interact with SE and also function as the central scaffold of NEXT. Loss of HEN2, RBM7 and simultaneous loss of both ZCCHC8A and ZCCHC8B results in the accumulation of miRNA precursors and miRNA maturation by-products, indicating that NEXT stimulates their degradation by the RNA exosome. Further support for a cooperation of SE and the NEXT complex in RNA surveillance is provided by the identification of common targets of SE and HEN2 by transcriptome-wide analyses. Interestingly, we observe that the accumulation of primary miRNAs caused by loss of HEN2 restores levels of mature miRNAs and mostly rescues the developmental phenotype of se-2 mutants. Taken together, our data suggest that SE is not only required for miRNA processing but also actively promotes the degradation of pri-miRNAs and other types of RNAs by recruiting the NEXT complex. We propose that SE fine-tunes miRNA biogenesis by facilitating miRNA processing or recruiting the NEXT complex to trigger the degradation of pri-miRNAs.Here, we show that Arabidopsis SE is associated with both CBC and NEXT hen2-2 T-DNA insertion line has been previously described (mtr3 and rrp4KD lines have been described in (http://arabidopsis.info/): zcchc8a-1 (SAIL_1230_H09), zcchc8a-2 (GK_598CO6), zcchc8b-1 (GK_667A12), rbm7-1 (SALK_005077), se-2 (SAIL_44_G12), hyl1-2 (SALK_064863). The double mutants zcchc8a-1 zcchc8b-1 (zch8a zch8b), hyl1-2 hen2-2, and se-2 hen2-2 were obtained by crosses. The transgenic line expressing FLAG-SERRATE under the control of 35S CaMV promoter in the se-1 mutant background has been described previously  and RBM7 (AT4G10110) including their putative promoter regions of 1600 and 1200 bp upstream of the transcription start sites, respectively, but lacking the stop codon and the 3′ UTR were cloned into vector pGWB604  and ZCCHC8A was fused to the C-terminal part of CFP (ZCCHC8A-cCFP) derived from vectors described in  encoding the Gal4 DNA binding and Gal4 activating domain, respectively. The vectors encoding MBP-SE and MBP-GFP have been described in -free protease inhibitor (Roche)). After removal of cell debris by centrifugation  the cleared supernatants were incubated for 30 min with anti-GFP or anti-FLAG antibodies coupled to magnetic microbeads . Beads were loaded on magnetized MACS separation columns equilibrated with lysis buffer and washed four times with 300 μl washing buffer . Samples were eluted in 100 μl of pre-warmed elution buffer (Milteny). Control IPs were performed in Col-0 using either GFP or FLAG antibodies.For each co-IP, 0.3 g of flowers pooled from 5http://proline.profiproteomics.fr/). The protein identification was validated using the following settings: Mascot pretty rank ≤1, False discovery rate (FDR) ≤ 1% for peptide-spectrum match (PSM) scores, FDR ≤ 1% for protein set scores. The total number of MS/MS fragmentation spectra was used to quantify each protein from at least two independent biological replicates. Biological replicates consisted of plants of the same genotype grown at different dates and in different growth chambers. In the case of ZCCHC8B we used plants derived from two independent transformants that were grown in parallel.Eluted proteins were digested with sequencing-grade trypsin (Promega) and analyzed by nanoLC-MS/MS on a QExactive+ mass spectrometer coupled to an EASY-nanoLC-1000 (Thermo-Fisher Scientific) as described before . Data weP-value corrected by Benjamini–Hochberg for each identified protein. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository  and the ribed in . Yeast cg, washed three times in buffer W5  and resuspended in 1 ml of 0.2 M mannitol, 15 mM MgCl2, 4 mM MES pH 5.7. A total of 100 μl of protoplasts were transferred to new round-bottom tubes containing 5 μg of the desired expression vectors. After adding 100 μl PEG/Ca2+ solution (40% (w/v) polyethylene glycol 4000 , 0.2 M mannitol, 100 mM Ca(NO3)2) samples were incubated 5 min at room temperature before the transformation was stopped by diluting the sample with 450 μl of buffer W5. Protoplasts were collected by centrifugation (3 min at 300 × g) and resuspended in 300 μl of buffer W1 . Protoplasts were incubated for 10–12 h at 22°C in dark before FRET-FLIM analyses and 18–22 h at 22°C before BiFC—FRET-FLIM experiments.The epidermal layer of Arabidopsis leaves was removed using the Tape-Arabidopsis Sandwich method . Leaves 4 photons per second for 90–120 s. PhoTime 64 (PicoQuant) software was used to calculate fluorescence decay curves for each pixel. The curves were fitted with the double-exponential decay model using default parameters. For each sample, more than 10 cells from at least three biological repeats (independent protoplast isolation and transformation) were used. The results were evaluated with the two-sided Mann–Whitney–Wilcoxon test .The PicoHarp300-Dual Channel SPAD system (PicoQuant) in combination with a Nikon A1Rsi microscope armed with the 40 × /1.25 water-immersion objective was used for FRET-FLIM analyses. The Picosecond Pulsed Diode Laser LDH-D-C-485 and Supercontinuum Tunable White Light Laser (488 nm) were used for generation of 100 ps excitation pulses at a repetition of 40 MHz. Images were of 256 × 256 pixels frame size and collected with an average count rate of around 10Escherichia coli as described before  following the protocol supplied by the manufacturer. Purified MBP-SE and MBP-GFP were bound to amylose beads (NEB) by overnight incubation at 4°C. Excess protein was removed by washing the beads three times with 20 mM Tris HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, cOmplete™ EDTA-free protease inhibitor (Roche) before the radiolabeled proteins were added. After incubation for 2 h at 4°C the beads were washed five times and proteins were eluted with 20 mM maltose. Eluted proteins were separated by 12% SDS-PAGE and detected using a FLA-5000 phosphoimager (Fujifilm).Recombinant SE and GFP proteins fused to maltose-binding protein (MBP) were purified from d before . [35S]-m18. Real-time quantitative PCR (qPCR) was performed using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and the QuantStudio 7 Flex Real-Time PCR System. GAPDH (AT1G13440) and PP2A (AT1G13320) mRNAs were used for normalization. Following log transformation, the Student's t-test  was used for statistical analyses. 3′ RACE-PCR was performed using the SMARTer PCR cDNA synthesis kit (Clontech) following the instructions supplied by the manufacturer. PCR products were separated by electrophoresis on a 1% (w/v) agarose/TBE gel and visualized by ethidium bromide. PCR products amplified from the hen2-2 samples were excised from the gel, cloned into pGEM, transformed in E. coli and analyzed by Sanger sequencing.For qRT-PCR and RNA blots, total RNA were prepared from rosettes of three biological replicates of four Figure  and fivehen2-2 with an average depth of 102 million mapped reads per library. The second RNA-seq experiment comprised WT, hen2-2, se-2 and se-2 hen2-2 with an average depth of 39 million mapped reads per library.Total RNA was prepared from rosette leaves from three biological replicates of four week-old plants grown at 22°C and 20°C for the first and second RNA-seq experiments, respectively. RiboMinus™ Plant Kit for RNA-Seq (Thermo Fischer Scientific) was used to deplete rRNAs. Sequencing libraries were prepared from 300 ng of ribo-depleted RNA  and indexed with NEB Next Illumina indexing primers (NEB). An Agilent Bioanalyzer with high-sensitivity DNA chip was used to confirm the quality of the libraries before they were sent for sequencing. Paired-end (2 × 150 bp) sequencing was performed at Fasteris, Geneva, Switzerland on an HiSeq4000 platform. The first RNA-seq experiment comprised wild-type (WT) and n = 3) RPM values. MIR genes structures are generated based on mirEX2 . RNA was quantified using the Qubit RNA Assay Kit (Life Technologies) and integrity was confirmed on the Agilent Bioanalyzer 2100 system. A total of 10 μg of each RNA sample were separated by electrophoresis on a 15% polyacrylamide 8M urea gel in 89 mM Tris, 89 mM borate, 2 mM EDTA. Small RNA fractions were cut out and purified from the gel. Libraries were prepared using the TruSeq Small RNA Library Preparation Kit (Illumina). Single-end (1 × 50 bp) sequencing was performed at Fasteris, Geneva, Switzerland on a HiSeq 4000 platform. Adapter sequences were removed from raw reads with FASTX-Toolkit (fastx_clipper). Bowtie was used to align clean reads to the Arabidopsis thaliana TAIR10 reference genome using command ‘bowtie –v 0 –k 1 –S’  fusion protein expressed in the ckground . The fusn Figure . Proteinn Figure  were then Figure . We alson Figure ,54–55. Sn Figure . CDC5 isn Figure . CDC5 inn Figure . Similarn Figure . In addin Figure . InteresWe then used Arabidopsis lines expressing GFP-tagged versions of ZCCHC8A, ZCCHC8B and RBM7 to test the association between SE and the NEXT complex. Indeed, SE was co-purified when ZCCHC8A, ZCCHC8B or RBM7 were used as baits Figure . This rein vitro pull-down assays  with fully annotated gene structures (i.e. including transcription initiation and termination sites) that are expressed from independent transcription units, i.e. not embedded in introns of protein coding genes. Of those 53 pri-miRNAs, 44 were detected in at least 3/6 libraries and had more reads in hen2-2 mutants as compared to WT plants   Figure .hen2-2 plants. The signals obtained with the primers specific to the pri-miRNAs (red bar-graphs) were 10-fold (pri-miRNA 159a) and 8-fold (pri-miRNA 161) higher in hen2-2 mutant plants. These results confirmed the results obtained by RNA sequencing and support the conclusion that hen2-2 mutants accumulate pri-miRNAs, in addition to the 5′ maturation by-products which were also observed in previous studies , snoRNAs (10.8%), snRNAs (1.4%) and other non-coding RNAs (1.4%) which likely represent direct targets of HEN2-mediated RNA degradation  and protein coding genes (88.2%) , MIR genes (10.8%), long non-coding (lnc) RNA (6.8%), ncRNA (2%), snRNA (1.4%) and snoRNA transcripts (6.1%)  Figure . The com) Figure . The majs Figure . Interess Figure , proteins Figure , and miRs Figure .se-2 hen2-2 samples as compared to hen2-2 or se-2 samples   . Using T-4) . Our datzcchc8a zcchc8b mutant. Hence, ZCCHC8A and ZCCHC8B have at least partially overlapping functions and can replace each other. Moreover, the duality of ZCCHC8A and ZCCHC8B genes is not systematically conserved in higher plants indicating that most species possess a single NEXT complex similar to metazoa.At last, a portion of Arabidopsis SE is associated with the NEXT complex, similarly to its human counterpart ARS2 . The assin vitro assays, ARS2 can directly interact with hMTR4 (An interesting observation is that the molecular interaction between NEXT and SE/ARS2 is differently mediated in human and plants. According to th hMTR4 , the funth hMTR4 . In addith hMTR4  and how se-2 hen2-2 is sterile while double hyl1-2 hen2-2 show normal fertility. Whether the sterility of se-2 hen2-2 is linked to the role of SE in RNA degradation which is not shared by HYL1 is unknown to date. However, although being caused by both direct and indirect effects, the massive perturbance of the transcriptome observed in se-2 hen2-2 highlights the importance of nuclear RNA surveillance when RNA processing and sorting processes, many of which do involve the SE/ARS2–CBC complex (se-2 hen2-2 double mutants. Our approach cannot discriminate between processing and degradation of snRNA transcripts, but the fact that levels of mature U6 snRNAs are not reduced in hen2-2, se-2 and se-2 hen2 would be in line with the idea that both HEN2 and SE are involved in the degradation of misprocessed or surplus transcripts rather than in snRNA production. Interestingly, human ARS2 was shown to stimulate the 3′ end processing of snRNAs, thereby preventing readthrough transcription (Based on the results presented here we propose that SE participates in RNA degradation through the recruitment of the NEXT complex. It is therefore interesting to note that the double mutant  complex ,34, are  complex ,59. An pcription . Hence, cription ,24,33,34hen2-2 and se-2 plants are truncated or misprocessed transcripts spanning the first intron (se-2 hen2-2 double mutants may also be explained by a dual role of SE in recruiting either splicing factors or NEXT.Another type of transcripts that we and other teams observed in t intron ,15,35,68t intron ,34,69. Ht intron , and a pt intron . A very hen2, rbm7 and zcchc8a zcchc8b mutants indicated that NEXT participates to the degradation of primary miRNA transcripts that are transcribed in excess. This conclusion is further supported by the observation that mutating HEN2 in se-2 and hyl1-2 plants, which have higher levels of precursors because dicing is slow and inefficient when SE or HYL1 are absent (pas2-2 mutant (hyl1-2 hen2-2 and se-2 hen2-2 mutants.Previous studies based on tiling microarrays already observed the accumulation of 5′ fragments excised from miRNA precursors upon downregulation of the RNA exosome or loss of HEN2 ,59. Our e absent ,4, has ae absent . We have2 mutant . The come-2 hen2-2 and hyl1-2 hen2-2 as compared with their respective single mutants can be explained with the contribution of HEN2 to pri-miRNA degradation. However, the interaction of SE with NEXT and the observation that se-2 mutants accumulate also typical targets of nuclear RNA-surveillance supports the hypothesis that the SE’s binding to pri-miRNAs can promote both, miRNA processing by recruiting the Microprocessor complex, or RNA degradation triggered by the recruitment of the exosome. Therefore, we propose that SE has a dual role in both miRNA biogenesis and pri-miRNA degradation.The observation that miRNA precursors accumulate to higher levels in both shttps://doi.org/10.6084/m9.figshare.c.4930314.v1.The RNA-seq data are available at GEO, accession GSE131542. The proteomic data supporting the conclusions of this study are available at PRIDE PXD014447. Uncropped high-resolution pictures of plants, gels and blots, qRT-PCR data, DESeq2 data and mapping files for the visualization of RNA-seq data are available at SERRATE: AT2G27100; ZCCHC8A: AT5G38600; ZCCHC8B: AT1G67210; RBM7: AT4G10110; HEN2: AT2G06990; HYL1: AT1G09700; PRP39A: AT1G04080.gkaa373_Supplemental_FilesClick here for additional data file.