We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm5C- and f5C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m5C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f5C sites on select tRNA. Finally, we show that f5C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m 5C) dioxygenase enzymes using an activity-based metabolic probing strategy. They reveal ALKBH1 as the major 5-formylcytidine (f5C) writer and characterize modification sites across mRNA and tRNA.Kleiner and co-workers profile RNA 5-methylcytidine (m Over 150 chemical marks have been identified on various types of RNA spanning all kingdoms of life4, but we still lack insight into the function and distribution of many modifications. A number of RNA modifications have also been implicated in human disease, underscoring the importance of investigating epitranscriptomic processes in biology6.Chemical modifications on central biomolecules play critical roles in biology. RNA is extensively modified by a diverse collection of post-transcriptional modifications (known as the “epitranscriptome”) that can affect RNA structure, base pairing, and protein-RNA interactions. In turn, these biochemical perturbations have been shown to control RNA metabolism, splicing, protein translation, and RNA localization, thereby affecting gene expression and higher-order physiological processes7. Therefore, specialized workflows relying upon modification-specific antibodies or selective chemoenzymatic transformations need to be developed for each modification, and as a consequence reliable mapping data is only available for a few RNA modifications7. Further, identifying relevant modification-associated enzymes and profiling their substrates is an additional obstacle and these enzymes are typically deduced based upon screening or homology to known enzymes.Studies of epitranscriptomic modifications in their native context typically require transcriptome-wide mapping together with characterization of writer, eraser, and reader proteins. These data can then motivate the generation and interrogation of functional hypotheses regarding the biological role of individual modifications. Modification mapping is a major challenge since many RNA modifications are “silent” in standard Next-Generation Sequencing (NGS) analysis and present at low stoichiometry5C). In eukaryotes, this modification is installed by NSUN/DNMT proteins on diverse RNA species10, and it can affect a broad range of cellular processes such as translation, stress response, mRNA nuclear-cytoplasmic trafficking, RNA stability, splicing, and DNA damage repair11. In addition, oxidation of m5C can generate hm5C and f5C in RNA15. Recent work has used antibody-based sequencing to map hm5C in mRNA and tRNA18, but the identity of hm5C writer enzymes has not been fully elucidated and we lack nucleotide-resolution sequencing for this modification. For f5C, transcriptome-wide sequencing in mammalian systems has not been reported, but mass spectrometry has revealed sites at the wobble position of cytosolic tRNA-Leu-CAA and mitochondrial (mt)-tRNA-Met generated by the Fe(II)/α-KG-dependent dioxygenase ALKBH120. Further investigation into these oxidized derivatives has been hampered by the lack of tools to confidently sequence these marks and to systematically profile the writers involved in their formation.One of the more abundant modifications found in cellular RNA is 5-methylcytidine . Our strategy relies upon metabolic labeling with the m5C-mimicking warhead 5-ethynylcytidine (5-EC)22, oligo(dT)-based RNA-protein enrichment, and quantitative mass spectrometry. Our results show that ALKBH1 is the major m5C-oxidizing enzyme in HEK293T cells. Further, we use nucleoside mass spectrometry to characterize the prevalence of hm5C and f5C on human RNA and show that human mRNA contains f5C modification. Finally, we develop 5-EC-aided individual-nucleotide CLIP sequencing (iCLIP) and pyridine-borane chemical sequencing of f5C to characterize hm5C/f5C sites generated by ALKBH1 on tRNA. We validate some of these sites orthogonally with Aldehyde Reactive Probe (ARP)-based sequencing and further propose a potential role of f5C in tRNA-Leu-CAA during translation. Taken together, our work expands the generality of RNABPP and deepens our understanding of oxidative m5C modifications in the human epitranscriptome.To address this gap, our group recently developed RNA-mediated activity-based protein profiling (RNABBP)5C RNA dioxygenases using RNABPP  based on the ability of its deoxynucleoside analog (5-EdC) to covalently label TET enzymes in an activity-based manner on DNA23. Further, 5-EC has been used for RNA metabolic labeling experiments to monitor RNA transcription22, similar to the more widely used 5-ethynyluridine (5-EU) nucleoside24. Therefore, we hypothesized that metabolically incorporated 5-EC would be able to react in an activity-dependent manner with RNA m5C dioxygenases  were largely unperturbed by 5-EC feeding , treated HEK293T cells, and measured incorporation by nucleoside LC-MS/MS and click chemistry imaging Fig. –2. We obing Fig. .26. For example, the single TET ortholog in Drosophila (dTet) is responsible for hm5C deposition in polyadenylated RNAs16. Moreover, TET1-CD can convert m5C to hm5C in RNA in vitro, and the catalytic domains of all three TET proteins, as well as full-length TET3, can induce hm5C formation in human RNA15. Gratifyingly, in cells treated with 5-EC and expressing TET1-CD, we observed the presence of a slower migrating band consistent with RNA-protein crosslinking -pulldown, but not the mutant version, or non-covalently bound TET1-CD in untreated cells . TET proteins are primarily DNA 5-methylcytosine oxidative demethylases, but they can also react with RNAP values (<0.05). We were pleased to find that four are known or putative RNA pyrimidine modifying enzymes  at the wobble position of mt-tRNA-Met20, and also reported to oxidize m5C at the wobble base of tRNA-Leu-CAA19. The activity of ALKBH1 on m5C sites in other RNA substrates is so far unknown. While ALKBH1 was not previously described to react with 5-EC, its identification using our method is consistent with its proposed catalytic mechanism28. We also identified m5C methyltransferases NSUN2 and NSUN5, and DUS1L, the mammalian homolog of yeast tRNA-dihydrouridine synthase (DUS) DUS1. NSUN-family m5C methyltransferases utilize a thymidylate synthase (TS)-like mechanism involving nucleophilic attack of a catalytic Cys residue on the C6 position of C to form an enzyme-bound intermediate, and a second Cys residue that mediates β-elimination after C5 methylation with SAM cofactor29. Interestingly, TS has been proposed to convert 5-ethynyl-2’-deoxyuridine into a thiol-reactive allene intermediate30. An analogous mechanism for NSUN proteins could lead to irreversible alkylation of the second Cys residue by 5-EC after rearrangement to its allene derivative  pulldown. We initially focused on ALKBH1 and TET2 since they have been shown to oxidize mlow Fig. , 8. For low Fig. . We coul6-methyladenosine (m6A) in RNA34 but with no reported activity on m5C. We observed substantially greater recovery of ALKBH1 over ALKBH5 with 5-EC  derived from the anticodon stem loop (ASL) of Mt-tRNA-Met .  instead of 5-EC was substituted in the reaction. Further, crosslinking was greatly reduced in reactions with 5-EC oligo 1 when α-KG, Fe(II), or RNA were omitted, as well as in reactions containing EDTA (iron chelator) or N-oxalyl glycine . Taken together, our data establish that 5-EC is an efficient mechanism-based probe for ALKBH1 that can be applied to crosslink RNA substrates in cells through metabolic labeling or for in vitro substrate analysis using chemically synthesized oligonucleotide probes.To understand the specificity of the ALKBH1 crosslinking, we compared the recovery of ALKBH1 against that of ALKBH5, a related Fe(II)/α-KG-dependent dioxygenase that can oxidatively demethylate N5C oxidation in cells. We prepared KO cells for each protein using CRISPR/Cas9 to target the corresponding gene in 293T cells  in RNA isolated from both wild-type (WT) and KO cells by quantitative nucleoside LC-QQQ-MS , which is common to tRNA  were increased ~4-fold relative to total RNA to 42.9 ± 0.9 ppm RNA fractions can be contaminated with modified nucleosides from more abundant RNA species35, given the low levels of f5C in total RNA and the purity of our poly(A) fraction  of both hmRNA Fig. –9. We coRNA Fig.  and 87–9RNA Fig. . We werells Fig. . In the RNA Fig. . In contppm Fig. . Next, wRNA Fig. . Taken t5C and hm5C formation. We found no statistically significant TET2-dependence of f5C levels  RNA and a 70% reduction in small RNA  are generated by ALKBH1-oxidation of NSUN3-dependent m5C residues, while the majority of f5C sites on small RNA originate from ALKBH1-oxidation of NSUN2-dependent m5C residues. Additionally, hm5C sites are generated primarily by ALKBH1-oxidation of NSUN2-dependent m5C residues. Further, we identify ALKBH1-dependent f5C sites on poly(A)RNA, broadening our understanding of the substrates of this enzyme and the distribution of f5C.The biosynthesis of hmRNA Fig. , indicat KO Fig. , 14, andion Fig. . While N42 previously reported that ALKBH1 can remove m1A on tRNA through oxidative demethylation and regulate translation. Therefore, we also investigated the role of ALKBH1 in cellular m1A levels by nucleoside LC-MS. While we observed no significant change in total m1A levels upon ALKBH1 knockout, m1A levels in small RNA increased by 68 ± 0.09%  indicated robust protein-RNA crosslinking  stop signatures  present in two independent biological replicates, (b) displaying a cDNA score >10 and (c) absent from the no 5-EC control OR present in the non-5EC control but with a 5-EC/control score ratio >1.5  corresponding to cytosolic tRNAs, indicating that tRNA is the major substrate of ALKBH1 Fig. . In contads Fig. , and we 5C in the anticodon or anticodon stem loop (ASL) of a number of cytosolic tRNAs. Consistent with crosslinking requiring 5-EC labeling, 21 of the identified tRNAs with crosslinks in this region have a C residue in the anticodon loop. We also observed 8 tRNAs with crosslinking peaks at positions 56–59 , which is likely due to the presence of a highly conserved m1A residue in the TψC -loop49. Rather than crosslinking to ALKBH1, this modification can induce an RT stop on its own50, thus generating an apparent crosslinking peak at that position. Another possibility is that the incorporation of 5-EC near this m1A residue could mediate crosslinking, particularly given the conservation of C at position 56 in many tRNAs. There was also an additional cluster of 10 tRNAs that displayed peaks at positions 25–28 . In addition to 5-EC-mediated crosslinking in this region, RT stops could be induced by the presence of a conserved N2,N2-dimethylguanosine (m22G) modification in this region51. Sequence motif analysis using multiple expectation maximizations for motif elicitation (MEME)52 revealed a tRNA consensus motif that matches the TψC-loop of various isoacceptors, likely due to peaks near m1A58 -based enrichment and were able to quantify ALKBH1-dependent f5C in mRNA, we also analyzed CLIP peaks mapping to non-tRNA regions. We found a total of 83 peaks, with 70.0% mapping to introns of protein-coding genes  hydroxylamine)53. Inspired by this method, we reasoned that we could also map f5C sites within RNA by ARP-based pulldown followed by NGS . In contrast, tRNA-Leu-CAA showed higher hm5C levels compared to f5C, consistent with previously reported oligonucleotide mass spectrometry analysis19. We further applied this approach to four other tRNAs from our 5-EC-iCLIP data – tRNA-Glu-CTC, tRNA-Gly-CCC, tRNA-Gln-CTG, and tRNA-Val-CAC. We were able to detect f5C but not hm5C in all substrates. For both tRNA-Glu-CTC . Interestingly, f5C levels in tRNA-Val-CAC and tRNA-Gln-CTG were unchanged upon ALKBH1 knockout 40, the pyridine borane-induced mutation rate is likely to be an underestimate of actual modification stoichiometry. In addition, we detected ALKBH1-dependent mutations in the anticodon of tRNA-Gly-CCC, tRNA-Val-CAC and Glu-CTC at a frequency of 0.95–4.3%  at position 25 of tRNA-Val-CAC and position 39 of tRNA-Gly-CCC, which require further investigation  was also comparable to that in the mt-tRNA-Met control (26%), with the exception of tRNA-Leu-CAA, which showed f5C/A levels at 15%. However, tRNA-Leu-CAA displayed the highest hm5C content post-oxidation  among all the substates. These higher levels of hm5C compared to f5C observed in vitro are consistent with the levels of modified nucleosides measured from the native tRNA  was not reliably detected above background after ALKBH1-mediated oxidation in vitro , a randomized 15-mer RNA library containing a single m5C site in the center (5)  , which suggests a slight preference of ALKBH1 for some linear substrates contained within. Taken together, our in vitro activity data shows that ALKBH1 can efficiently oxidize m5C-containing stem loops taken from diverse tRNAs. Further, in select sequence contexts, ALKBH1-mediated m5C oxidation can also occur on linear substrates.In addition, we measured the activity of ALKBH1 on linear substrates including mutated mNA-Met 3–, a rando–8) Fig. –21. Whilogo Fig. . The m5C5C at the wobble position of tRNA Leu-CAA, we posited whether the modification could facilitate translation by decoding non-cognate TTA Leu codons, analogous to the proposed function of f5C on mt-tRNA-Met in decoding both AUA and AUG codons. We assessed the translation efficiency of TTG and TTA codons through an in-cell dual-luciferase assay in WT 293T or ALKBH1 KO cells. In this assay, cells are transfected with a construct containing Renilla luciferase (Rluc) linked to Firefly luciferase (Fluc). Efficient readthrough of the linker, which, in this case consists of a string of either TTG, TTA, or random codons can be quantified by an increase in Fluc signal as compared to the Rluc output. Under normal cell culture conditions, we observed no significant changes in fluorescence among different constructs or cell lines  in 5-EC treated samples, which suggests that this probe does not significantly affect the cellular activities of m5C-forming or m5C-oxidizing enzymes and hints to its broad applicability in other settings. Analogous approaches with alkyne-containing metabolites should be readily applicable to study diverse oxidative enzymes acting upon biopolymer and small molecule substrates, as well as to study other proposed substrates of ALKBH162.We chose 5-EC as a mechanism-based probe for RNA m5C substrates for ALKBH1-mediated oxidation to f5C. Previous studies have reported two RNA m5C sites that are substrates for ALKBH1—m5C34 in mt-tRNA-Met (oxidized to f5C) and m5C(m)34 in cyt-tRNA-Leu-CAA (oxidized to hm5C/f5C and hm5Cm/f5Cm). Using nucleoside LC-MS analysis of fractionated RNA from WT, ALKBH1 KO, NSUN2 KO, and NSUN3 KO cells, we show that ALKBH1 installs f5C on the poly(A)RNA fraction through oxidation of NSUN2- and NSUN3-dependent m5C sites. The pathway for f5C formation on C34 of mt-tRNA-Met requires NSUN340 and CLIP studies have proposed that this is the only substrate for the enzyme;63 however, we observe no NSUN3-dependent depletion in f5C levels on small RNA (which contains mt-tRNA-Met), and instead observe NSUN3-dependent depletion of f5C in total RNA and poly(A)RNA, further supporting the existence of f5C (and m5C) sites catalyzed by NSUN3 and ALKBH1 beyond the canonical mt-tRNA-Met substrate. One important consideration that must be accounted for in the interpretation of low abundance hm5C and f5C sites is the possibility that these are a result of catabolism of abundant hm5C/f5C-containing RNAs followed by non-specific incorporation during transcription. While an analogous chain of events has been proposed to explain the presence of 6-methyladenine on mammalian DNA64, we do not favor this scenario since hm5C and f5C are present at low overall abundance in the transcriptome (in contrast to m6A), and it is unknown whether they are efficiently processed by pyrimidine salvage pathways.Our work identifies new RNA m5C/f5C sites at nucleotide resolution, we developed and applied multiple complementary sequencing platforms. 5-EC-iCLIP analysis and ARP-sequencing profiled ALKBH1 m5C substrates transcriptome-wide and showed that its major substrate is tRNA, which is consistent with a previous UV-CLIP study for ALKBH142, but we also found additional minor modification sites on intronic mRNA, UTR, lncRNA, and CDS regions. Indeed, the crosslinking peak at the wobble base of cyt-tRNA-Leu-CAA was the most abundant of all those identified and is likely the major ALKBH1 m5C substrate in addition to mt-tRNA-Met. The ability of ALKBH1 to act upon both nuclear/cytosolic and mitochondrial substrates is worthy of further exploration due to the primarily mitochondrial localization of the protein65. In addition, we find 5-EC-mediated crosslinking of ALKBH1 to 38 other tRNA isodecoder famlies, with the majority of peaks mapping to the ASL, and ARP-sequencing supports additional tRNA substrates beyond cyt-tRNA-Leu-CAA and mt-tRNA-Met. We interrogated the presence of f5C at these sites using targeted RT-PCR and pyridine borane-mediated f5C-to-D transformation (thereby producing characteristic C-to-U mutations upon RT)54. While not all 5-EC-iCLIP peaks in tRNA could be validated by pyridine borane sequencing, this may be due to the presence of hm5C (which does not react with pyridine borane), or due to crosslinking mediated by 5-EC incorporation at sites that lack m5C in the native system. In the latter case and supported by our comprehensive in vitro biochemical studies with recombinant ALKBH1, our analysis supports promiscuous activity of ALKBH1 on a variety of tRNA ASL sequences and even some linear m5C-containing sequences, suggesting that hm5C and f5C installation is likely to occur at these sites in systems where m5C deposition is upregulated.To map ALKBH1-dependent hm5C in vivo and in vitro to hm5C23. Our data do not implicate TET2 in the formation of f5C in human total RNA, but do show that TET2 contributes to global hm5C formation , consistent with previous reports66. We propose that 5-EC metabolic labeling could be applied in diverse biological contexts to map the substrate of TET2 in an activity-dependent manner using 5-EC-iCLIP or analogous platforms.We also identify TET2 as a 5-EC reactive protein, consistent with previous studies showing that TET2 converts RNA m5C. The presence of f5C at the wobble base of mt-tRNA-Met has been proposed to facilitate reading of unconventional mitochondrial Met codons67, and ALKBH1 KO leads to mitochondrial dysfunction63. The role of ALKBH1 in mediating cytoplasmic translation has been suggested to occur through demethylation of tRNA m1A5842; however, it is plausible that ALKBH1-dependent installation of f5C or related derivatives in the ASL of cytoplasmic tRNAs may also serve to tune decoding functions or to regulate tRNA structure and/or stability. Our initial studies of wobble f5C in tRNA Leu-CAA suggest a role for the modification in decoding non-cognate codons more efficiently under stress and could implicate f5C in the regulation of stress response, but more research is needed in this area. Additionally, f5C modifications in mRNA, if present at substantial stoichiometry, may modulate the expression of individual protein products. Continued work in this area will undoubtedly broaden our appreciation of epitranscriptomic processes and RNA biology.Finally, our work motivates further investigation into the biological role of ALKBH1 and fPlasmids encoding cDNA were from the following sources: TET1 (Addgene #49792), TET1-CD H1762Y A1674A (Addgene #84479), TET2 (Addgene #41710), ALKBH1 (Genscript #OHu05179D), MPP8 (Dharmacon #MHS6278-202829247). For transfection and Flp-In cell line construction, the full sequences of TET2, ALKBH1, MPP8, TET1(CD) Mut, and the catalytic domain (CD) of TET1 (aa 1418-2136) were cloned into a modified pcDNA5/FRT/TO vector containing an N-terminal 3xFLAG tag. For protein expression, ALKBH1 was cloned into pGEX-6P-1. For the generation of KO cell lines, DNA oligos coding for guide RNA (gRNA) sequences were cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 . The oligos were phosphorylated with T4 PNK  and ligated with T4 DNA ligase  into a pX330 backbone digested with BbsI-digested . For the dual-luciferase assay, reporter plasmids were generated by overlap PCR from pcDNA3 RLUC POLIRES FLUC (Addgene #45642) and ligation into pCDNA3.1 (Invitrogen). TTA reporter plasmid, TTG reporter plasmid, and random reporter plasmid were obtained by subcloning TTATTATTATTATTA, TTGTTGTTGTTGTTG, or AGCCCCGGGGAGCTC linkers, respectively, between the Renilla luciferase (RLuc) and Firefly luciferase (FLuc) portions.l-glutamine (Life Technologies). For stress treatments when applicable, cells were heat-shocked at 42 °C for 1 h, treated for 1 h with 0.5 mM NaAsO2 (Sigma), treated for 4 h with 0.2 mM NaAsO2, or grown for 8 h in FBS- and glucose-free DMEM supplemented with 5 mM glucose (Gibco).Wild-type (WT) HEK 293T, and Flp-In TRex 293 cells were grown at 37 °C in a humidified atmosphere with 5% CO2 in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (Atlanta), 1x penicillin-streptomycin (Gibco Life Technologies) and 2 mM 6 cells per well in six-well plates, and co-transfected with the appropriate pCDNA5/FRT/TO-3xFLAG-tagged plasmid (0.2 µg) and pOG44 plasmid . After selection with 100 µg/mL hygromycin B and 15 µg/mL blasticidin, colonies were expanded. To confirm expression of the desired protein, cells were induced with tetracycline (0–1 µg/mL) for 24 h. Cells were harvested and lysed in NP-40 lysis buffer . Protein concentration was normalized to 2 mg/mL, and proteins were separated by SDS-PAGE and analyzed by western blot with anti-FLAG M2 antibody .To generate stable cell lines expressing 3xFLAG-tagged ALKBH1, TET2, and MPP8, Flp-In TRex 293 cells were seeded at 0.6 × 106) were seeded in a six-well dish. After 24 h, pX330 plasmid containing the gRNA for the target protein (2 µg) and pcDNA3-FKBP-EGFP-HOTag3  were co-transfected using Lipofectamine 2000 (Thermo Scientific). Cells were FACS-sorted 48 h after transfection. The top 95% of cells with green fluorescent protein (GFP) signals were sorted as single cells into 96-well plates. Surviving colonies were expanded to 6-well plates and then 10-cm plates. KO was confirmed by genomic PCR and western blot with mouse β-actin antibody  and either rabbit anti-ALKBH1 , anti-TET2  or anti-NSUN2  antibodies.HEK293T WT cells (0.8 × 10HEK293T WT cells were seeded in 96-well culture plates (4000 cells in 200 µl of medium per well) on day 0. On day 1, cells were treated with 5-EC in DMSO at the final concentrations of 0, 10, 50, 100, 200, 500, 1000, 2000, and 5000 µM. Cell viability was measured using the MTS assay  at 12 h, 24 h, 36 h, and 48 h post-treatment following the manufacturer’s instructions. The absorbance of each well was read at 490 nm on a Synergy H1 Microplate Reader (BioTek). All treated cells were normalized to the 0 µM sample and the curve was fitted based on a 4-parameter dose-response equation using GraphPad Prism:Three independent biological replicates and three technical replicates were analyzed.6 cells per well in a 6-well plate. Cells were allowed to grow 18–24 h and were treated with 1 mM 5-EC or vehicle for 4 h. After labeling, media were removed, and the coverslips were washed with PBS three times. Cells were fixed for 20 min at room temperature in PBS containing 3% paraformaldehyde and 2% sucrose adjusted to pH 7.3 and washed with PBS 3 times for 5 min each time. Cells were permeabilized with PBST (PBS + 0.1% Triton X-100) for 20 min at room temperature and washed 3 times with PBS for 5 min each time. Cellular RNA was labeled by Cu-assisted azide-alkyne cycloaddition (CuAAC) by incubating each coverslip upside-down (cell-side down) onto droplets (100 µL) of “click” solution  for 2 h at room temperature in the dark. Coverslips were washed briefly 3 times with PBST and then rewashed more thoroughly 5 times with PBST for 10 min each time to remove any remaining dye. Nuclei were stained with Hoechst 33342  in PBS for 5 min, and coverslips were washed with PBS twice for 5 min. Coverslips were mounted on cell-face down in ProLong Gold AntiFade Reagent (Life Technologies) atop a microscope slide and fixed in place with nail polish. Fixed cells were imaged on a Nikon Eclipse Ti microscope equipped with a 100x objective, a CMOS camera, and the NIS Elements AR software. The TRITC channel was used to excite the Cy3 dye. Images used for direct comparison were acquired using standardized illumination and exposure parameters and presented with identical LUT settings.WT 293T cells were seeded on a round glass coverslip at 0.6 × 106 cells/well in six-well plates and transfected with the pcDNA5-TET1(CD) plasmid (2 μg) and Lipofectamine 2000  following the manufacturer’s instructions. Twelve (12) h post-transfection, cells were treated with fresh media containing 5-EC or 5-EU (0.2–1 mM) for 16 h. Cells were harvested and lysed in NP-40 lysis buffer and incubated on ice for 30 min. The lysate was spun at 21,000 × g at 4 °C, normalized to 2 mg/mL protein, run on a 10% SDS-PAGE gel, and analyzed by western blot (anti-FLAG) as described above.WT 293T cells were seeded at 0.6 × 105C in Supplementary Fig. 69. 1 mL of oligo(dT) lysis buffer  and 75 µL of equilibrated oligo(dT) beads were used per plate. Lysis, enrichment, and washes were performed at ambient temperature, and washes were performed with lysis buffer, NP-40 washing buffer , and NP-40 free washing buffer (just for proteomics experiments). Bead-bound RNA was eluted with oligo(dT) elution buffer  for 5 min at 80 °C with shaking. The incubation-wash-elution procedure was repeated 2 more times, and the elutions combined and concentrated to ~75–50 µL. RNA in the elutions was digested with 25 unit/mL RNase A and 2000 unit/mL RNase T1 in 40 mM NaCl and 2 mM MgCl2 at 37 °C for 1 h. The remaining protein and the input were run on a 10% SDS-PAGE gel analyzed by western blot (anti-FLAG). Experiments were performed in triplicate.Flp-In TRex 293 cells expressing the desired protein  or WT HEK 293 T cells (for proteomics experiments) (5–10 × 10 cm plates) were grown to 50% confluency, induced with tetracycline (1 µg/mL), and cultured for 24 h to ~80% confluency before overnight treatment with 1 mM 5-EC (500 mM stock in DMSO) or vehicle  as described previously71. Data were analyzed using Scaffold  to validate MS/MS-based peptide and protein identifications. Quantitation of protein abundance was done by spectral counting. Proteins were only selected for quantification if they were present in at least two replicates and not in the negative control.Sample volume was adjusted to 200 µL with 50 mM Ammonium Bicarbonate pH 8 and incubated with 5 mM TCEP at 60 °C for 10 min and then with 15 mM chloroacetamide in the dark at RT for 30 min. Proteins were digested with Trypsin Gold  and incubated at 37 °C for 16 h. Samples were acidified 0.2% TFA and desalted using SDB stage-tips72 and small RNA depletion (with Zymo Clean and Concentrator 5. RNA samples (3 µg) were digested in 20 µL with 2 units of Nuclease P1 (Wako) and final concentrations of 7 mM NaOAc and 0.4 mM ZnCl2 at 37 °C for 2 h. The digested mixture was subjected to dephosphorylation in a total volume of 30 µL with 2 units of Antarctic Phosphatase  and a final concentration of 1X AnP buffer at 37 °C for 2 h. Quantification of ribonucleosides was done an Agilent 1260 LC Infinity II system coupled to an Agilent 6470 LC/TQ module as reported previously21 and with a separation gradient from literature precedence73. Analyte detection was done by dynamic multiple reaction monitoring (DMRM) with the parameters in Table 5C, 5-EC, hm5C, f6C, i6A) were analyzed in 100–200 ng. Commercial nucleosides were used to generate standard curves. Relative levels of m5C, 5-EC 5-EU, hm5C, f6C, and i6A were calculated by normalizing the concentration of modified nucleoside to that of the respective canonical nucleoside.Total RNA was extracted using TRIzol reagent following the manufacturer’s indications. Small RNA was isolated from total RNA using the adjusted protocol from RNA Zymo Clean and Concentrator 5 (Zymo Research). mRNA was isolated by 2 rounds of poly(A) pulldown as described above and further subjected to ribo-depletion with custom synthesized probes complementary to human rRNAMetabolic incorporation of 5-EC was performed by feeding WT 293T cells at ~80% confluency with 1 mM 5-EC (500 mM stock) or vehicle in fresh DMEM for 16 h. RNA was processed for QQQ LC-MS/MS quantification on Agilent MassHunter Workstation Data Acquisition version 10.0 as described above using relevant standards . Lysates were treated with Turbo DNase (Thermo) and a low (1:200) or high (1:50) concentration of RNase I (AM2295) at 37 °C for 3 min with rotation, after which they were centrifuged at 20,000 × g for 15 min at 4 °C. The supernatant was incubated with anti-FLAG beads  at 4 °C overnight, and the immunoprecipitated material was washed three times with high-salt buffer and then once with CutSmart buffer (NEB). The beads were treated with Quick-CIP (NEB) at 37 °C for 30 min with rotation, washed twice with high-salt buffer, and then incubated with pre-adenylated and biotin-labeled L3 linker by using T4 RNA ligase I (NEB) at 16 °C overnight. The beads were boiled and analyzed by Western blot for biotinylation with the chemiluminescent nucleic acid detection module (Thermo). The rest of the sample was gel purified based on the position of the biotin signal by using D-tube dialyzer midi (Merck-Millipore). Proteinase K-digested and purified RNA was used for reverse transcription with oligos containing randomized barcodes (UMIs) and two inversely oriented adaptor regions separated by a BamHI restriction site. cDNAs were size-purified  on TBE-Urea gels and then circularized by CircLigase II (Epicentre). Circularized cDNAs were digested with BamHI and linearized cDNAs were amplified using Solexa primers and submitted for Illumina sequencing.Library preparation for iCLIP was adapted from the literaturehttps://imaps.genialis.com/iclip). Briefly, random unique molecular identifiers were used to distinguish and discard PCR duplicates, and adaptor/barcode sequences were removed. Trimmed reads were mapped to tRNA/rRNA with STAR (v.2.7.0f). The unmapped reads were further mapped to GRCh38 with STAR (v.2.7.0f) and only uniquely mapping reads were used for further analysis. For iCLIP: iCount generated raw crosslinking sites were used for peak calling analysis by Paraclu with following parameters: Minimal sum of scores inside a cluster:10; Maximal cluster size: 6; Minimal density increase:1. For ARP pull down: peaks were called using Paraclu with default parameters. Only peaks present in two replicates and unique to the 5-EC-treated  sample or showing a fold-change of greater than 1.5 (treated versus input) were kept after intersection. Sequences 5 bases upstream and downstream were extracted from the reference and used for motif analysis by MEME (V.5.3.3) using the default parameters.The iCLIP and ARP pulldown sequencing data was processed using iCount Primary Analysis (Consensus mapping) pipeline on iMAPs web server  for 18 h. Lysis by sonication was performed in buffer containing 1X TBS, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.2 mg/mL lysozyme (Thermo), and 1% Triton X-100, supplemented with 1 mM PMSF, protease inhibitor tablet, and Benzonase (Sigma). The lysate was purified using Pierce glutathione agarose resin (ThermoFisher) following the manufacturer’s instructions. The GST tag from promising fractions was cleaved with PreScission protease  while simultaneously dialyzing in 1x TBS at 4 °C overnight. Cleavage was confirmed by SDS-PAGE, and the free GST tag was removed by running the sample over fresh or regenerated glutathione resin and collecting the flow-through. The protein was further fractionated on a MonoQ 5/50 GL anion exchange column  on a gradient of 50 mM to 1 M NaCl (in 50 mM Tris HCl pH 8) for 20 column volumes. The most concentrated fractions were combined dialyzed overnight at 4 °C in 50 mM Tris HCl pH 7.5 and concentrated to at least 1 mg/mL.The sequence-verified construct pGEX-6P-1-ALKBH1 was transformed into 5C-containing RNA library (oligo 5), a mix was created by combining each TBDMS-protected RNA phosphoramidite  in the ratios previously reported74. Oligos 3–4 and 6–8 were cleaved from the resin and 2’OH-deprotected and butanol-precipitated under standard conditions.Oligonucleotide synthesis was done on an ABI 394 oligonucleotide synthesizer (Applied Biosystems) using standard coupling conditions and commercial oligosynthesis reagents and phosphoramidites (Glen Research) unless otherwise noted. For synthesis of the random m1–2 were synthesized with 5-EC phosphoramidite  or m5C phosphoramidite (Glen Research), respectively, using ultra mild phosphoramidites (Glen Research). The final product was manually detritylated one last time with 3% dichloroacetic acid on the instrument until the orange coloration observe in the lines (from the trityl cation) disappeared. The oligos were cleaved from the resin in a solution of 3:1 NH4OH/EtOH at rt for 24 h with rotation, and the supernatant was dried on speedvac. 2’-OH deprotection was performed on the residue with 1 M TBAF on THF (1 mL) at rt for 18 h with rotation. The solution was diluted with THF (1 mL), desalted on NAP-10 columns , eluted with 2.5 mL DEPC-H2O, and lyophilized. Incompletely lyophilized samples were precipitated with 1 mL EtOH + 100 μL 3 M NaOAc pH 5.2, dried via speedvac, resuspended in H2O, and re-lyophilized.Oligos 5 were purified by reverse-phase HPLC on a Zorbax Eclipse XDB-C18 semiprep column (Agilent) using a gradient of 5 to 40% acetonitrile in 0.1 M triethylammonium acetate over 50 min or 0 to 20% acetonitrile over 30 min and characterized by ESI-TOF HRMS and MALDI-TOF MS  was reacted with 0.1 µM ALKBH1 in a 250 µL reaction containing 20 mM HEPES pH 6.8, 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 4 mM ascorbic acid, 100 µM 2-oxoglutarate, and 80 µM ammonium iron (II) sulfate. The mixture was incubated at 37 °C for the time indicated in Fig. 5C and generation of hm5C and f5C were measured in 6.25 pmol of the sample by LC-QQQ-MS using commercial standards. Experiments were performed in triplicate.ALKBH1 enzymatic assays were carried out as reported previously2, 1 mM alpha-ketoglutarate, 100 μM FeCl2, 1 μM RNA oligo, and 0.2 μM recombinant ALKBH1. The reactions were incubated for 1 h at 37 °C and quenched with the addition of 1 μM 0.5 M EDTA or 10 μL 3x SDS sample buffer for Western blotting. Crosslinked protein was separated on 12% SDS-PAGE and Western-blotted for ALKBH1 as indicated above.Crosslinking reactions were prepared in 20 μL with the following concentrations of reagents: 50 mM tris pH 8, 50 mM KCl, 2 mM ascorbic acid, 1 mM MgCl42. Small RNA was extracted from WT or ALKBH1 KO 293 T cells using the small RNA enrichment protocol from Zymo RNA Clean and Concentrator-5 (Zymo). Depending on tRNA abundance and probe efficiency, the pulldown was done on 5–25 µg of bulk small RNA. Streptavidin M-280 magnetic beads (Invitrogen) (10 µL bead per 5 µg RNA input) were used. RNA and probe-conjugated beads were incubated at 42 °C for 3 h. Elution was performed twice at 75 °C for 5 min in 10 mM Tris pH 7.5.Specific tRNAs were enriched by antisense pulldown with biotin-labeled antisense probes in Table 75. RNA was transferred overnight to a Biodyne B Nylon membrane (Thermofisher) and immobilized under a 254 nm UV lamp for 10 min. Blocking was done in pre-hybridization buffer  supplemented with 50 µg/mL of salmon sperm DNA for 2 h at 42 °C. Annealing was done in pre-hybridization buffer for 4 h at 42 °C with 50 nM of the corresponding biotinylated probe  and 1 M pyridine borane for 16 h at 37 °C in water. The product was purified on Zymo-Spin columns. 100 ng reacted RNA was incubated with 10 μM RT primer and 1 μL 10 mM dNTP mix at 65 °C for 5 min. The reaction was reverse transcribed with Superscript II following the manufacturer’s instructions. cDNA was then PCR amplified by AccuPrime™ Pfx DNA Polymerase (Thermo). The PCR products were gel purified and submitted for high-throughput amplicon sequencing (Genewiz).5C sites.The reads were first mapped to tRNA reference sequences by STAR . Only uniquely mapped reads were kept for further analysis. Pileup files were generated using Samtools mpileup  with default parameters. The variants were called using VarScan mpileup  with the default parameters.Mutations that were only present in WT samples but not ALKBH1 KO samples and control samples were identified as ALKBH1-dependent f53. Briefly, 100 ng f5C containing oligo was first added into 100 μg fragmented RNA, the mix was then incubated with 2 mM ARP in 40 mM aqueous NH4OAc buffer pH 5.0 supplemented with 100 mM anisidine for 24 h at 25 °C. RNA was then purified using phenol/chloroform extraction. 200 ng RNA was taken out as input, and the rest of the samples were incubated with 50 μl pre-washed streptavidin beads in 400 μl IP buffer  for 2 h at 4 °C. The beads were then washed twice with IP wash buffer . The beads and input samples were treated with Quick-CIP (NEB) at 37 °C for 30 min with rotation, input samples were purified using RNA Zymo Clean kit (Zymo Research), and beads were washed twice with wash buffer. Input RNA and bead were then incubated with pre-adenylated L3 linker (rAppAGATCGGAAGAGCGGTTCAG/ddC/) by using T4 RNA ligase I (NEB) at 16 °C overnight. On-bead reverse transcription was performed using Superscript III with iCLIP RT primers . cDNA was eluted using RNaseH and then size-purified (60–100 nt and 100–200 nt) on TBE-Urea gels. Extracted cDNA was circularized by CircLigase II (Epicentre). Circularized cDNAs were digested with BamHI and linearized cDNAs were amplified using Solexa primers and submitted for Illumina sequencing.Total RNA extracted from WT or ALKBH1 KO cells was first treated with 2 μl DNase I (NEB) to remove DNA contamination. The total RNA was then fragmented into 150 nt fragments using RNA Fragmentation Reagents (Thermo). The incubation of fragmented RNA samples with Aldehyde Reactive Probe (ARP) was carried out with some modification from a previous reportp < 0.05 and **p < 0.01.For the translation assay, 500 ng of each reporter plasmid were transfected with lipofectamine 2000 into WT HEK293T or ALKBH1 KO cells in a 24-well plate. After 4 h, cells were washed with PBS and changed to glucose deprivation medium  or normal medium  and grown overnight. Cells were harvested in passive lysis buffer (Promega) following the manufacturer’s protocol. The assay was performed using Dual-luciferase Reporter Assay (Promega) following instructions from the manufacturer. Luciferase signal was measured in a Synergy H1 Microplate Reader (BioTek). Fluc signal was normalized to luciferase Rluc signal to evaluate the translation efficiency of each reporter. Three independent biological replicates were analyzed and a student T-test was applied with *Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Long RNAs vary extensively in their post-transcriptional fates, and this variation is attributed in part to short sequence elements. We used massively parallel RNA assays to study how sequences derived from noncoding RNAs influence the subcellular localization and stability of circular and linear RNAs, including spliced and unspliced forms. We find that the effects of sequence elements strongly depend on the host RNA context, with limited overlap between sequences that drive nuclear enrichment of linear and circular RNAs. Binding of specific RNA binding proteins underpins some of these differences—SRSF1 binding leads to nuclear enrichment of circular RNAs; SAFB binding is associated with nuclear enrichment of predominantly unspliced linear RNAs; and IGF2BP1 promotes export of linear spliced RNA molecules. The post-transcriptional fate of long RNAs is thus dictated by combinatorial contributions of specific sequence elements, of splicing, and of the presence of the terminal features unique to linear RNAs. Ron and Ulitsky found using massively parallel assays that the effects of short RNA sequences on the subcellular localization of their host RNAs are strongly dependent on the host RNA form, linear or circular, and spliced or unspliced. Nuclear export of long RNAs is generally thought to be dependent on factors that recognize a 5′ cap3. Surprisingly, circRNAs also appear to mostly localize to the cytoplasm5, but there are also examples of circRNAs which function in the nucleus7. It is not known if the cytoplasmic presence of circRNAs results from an active export process or from their high stability9 that allows their escape into the cytoplasm during mitosis in the course of cell division. The function of the vast majority of circRNAs remains unknown10, and it is unclear how many circRNAs are functional12, but it is likely that those that do carry out influential functions require specific subcellular distribution patterns. Furthermore, it is possible that aberrant circRNA accumulation, which may occur during aging or neurodegeneration14, can have localization-dependent detrimental effects.Eukaryotic cells are divided into two main compartments: the nucleus where RNAs are made and processed, and the cytoplasm where some RNAs are translated into proteins. Only a subset of the RNA molecules that are made are efficiently exported from the nucleus to the cytoplasm , other RNA molecules, including many lncRNAs, are enriched in the nucleus15, it was shown that circRNAs are widely distributed among different subcellular fractions , and that circRNAs in the different compartments had different characteristics in terms of length and G/C content. In addition, perturbations of different export factors in Drosophila DL1 and human HeLa cells have shown that the lengths of mature circRNAs dictate the mode of their nuclear export16. In neurons, some circRNAs were shown to localize to synapses17, but the elements that dictate such localization are unknown.Beyond the nuclear and the cytoplasmic compartments, very little is known about localization of circRNAs to other compartments. In a recent study which systematically studied circRNA representation in subcellular fractions of HepG2 cells20, and that splicing factors can facilitate nuclear export by recruitment of export factors to the mature mRNA21. It is unknown if the splicing involved in formation of circRNAs contributes to their export.During splicing, introns are removed from the pre-mRNAs by the spliceosome to generate the mature mRNA. Splicing and polyadenylation of the pre-mRNA are typically rapid and mostly co-transcriptional, and are carried out by proteins which sometimes remain bound to the processed RNA. By comparing the localization and quantity of spliced and unspliced RNAs in the different cell compartments it was shown that splicing enhances the export of RNAs from the nucleus to the cytoplasmtrans and cis-regulatory factors. Massively parallel RNA assays (MPRNAs) were recently introduced as a tool to study the contribution of short cis-acting sequences to retention of lncRNA in the nucleus or on chromatin, or to efficient export of single-exon RNAs25. All these studies were based on an unspliced ‘host’ mRNA carrying the variable sequences in its 3′UTR. Cells expressing the inserts were fractionated into chromatin, nuclear or cytoplasmic fractions, and the fragments from each compartment were sequenced. Oligos sequences that are enriched in one of the compartments were considered to drive export or nuclear retention. There was some overlap between the identified motifs23, but many motifs were observed in one study but not in others, possibly due to the differences in their experimental design. A key question that we set out to systematically address here is what is the contribution of the host RNA sequence and form to the effect that short sequence elements have on RNA localization and its stability.The localization of long RNAs in the cells is regulated by 26 on nuclear and cytoplasmic fractions  Unspliced RNA—produced from the β-globin-Δintrons expression vector27, which contains the human β-globin gene where both introns were removed, and therefore it is not spliced. The RNA produced from this vector was previously shown to be much less efficiently exported than WT spliced β-globin mRNA28  ; we cloned CircLibA and NucLibA libraries into expression vectors that produce three different RNA forms Fig. : (1) LinA28 Fig. . NucLibAT1) Fig. . These brelative changes when compared with the baseline localization of each backbone  ratios of the library were quantified by qRT-PCR alongside the GAPDH and MALAT1 transcripts to evaluate fractionation efficiency  and between the two circular forms , than between the circular and the linear forms . In order to identify common features in tiles that are localized to the different compartments, we compared tiles that were significantly enriched in the cytoplasmic (log2(Nuc/Cyto) <−0.3, roughly corresponding to a 25% difference, and P < 0.05) or in the nuclear (log2(Nuc/Cyto) > 0.3 and P < 0.05) fractions of each library , while in the circular form there is almost no difference between these groups  also likely underlie the slightly different distributions of Nuc/Cyto ratios for tiles derived from the two libraries  to inhibit transcription following plasmid transfection, extracted RNA at different time points, and sequenced the tiles. Then, we used the expression levels to calculate the half-life time of each fragment at each context. Since we observed high correlation between the Nuc/Cyto ratios of tiles in the two circRNAs contexts (circPVT1 and SCRcircPVT1), the following experiments were conducted using only circPVT1 expression vectors.Short sequence elements can also affect host RNA stability36, and among the linear transcripts, those unspliced tended to be relatively unstable  each of them in MCF-7 cells Fig.  and perf41, has roles in RNA splicing42, and was reported to have a function as an export adapter that is involved in mRNA nuclear export through the NXF1/TAP pathway43. Tiles bearing the SRSF1 binding motif were enriched in the nuclear fraction especially when expressed in either of the circular backbone sequences  restricts circRNA expression in DrosophilaIn order to evaluate the transcriptome-wide effect of SRSF1 on circRNA localization, we used RPAD to characterize circRNA expression in the nucleus and cytoplasm of MCF-7 cells following transfection of siRNAs targeting SRSF1 or a control. As expected from our MPRNA results, presence of SRSF1 binding motifs in the circRNA sequence was associated with increased nuclear localization in control cells , with much larger effect sizes in the unspliced transcripts—transcripts from unspliced genes with >=2 SAFB eCLIP clusters in HepG2 cells were 2.8-fold more nuclear than those without SAFB eCLIP clusters, compared to just 1.47-fold for spliced genes , or Zipcode-binding protein (ZBP)) are a family of conserved RBPs that regulate a large repertoire of mRNA transcripts through diverse mechanisms51, we examined the association between IGF2BP1 binding and RNA levels and the stability of tiles. Tiles with the IGF2BP binding motif were significantly less stable in the spliced context compared to the rest of the tiles   Fig. . This sust) Fig. , suggestR = 0.6–0.79 between replicates). IGF2BP1 KD led to an increase in the nuclear enrichment of RNAs containing increasing numbers of IGF2BP1 eCLIP binding clusters . We also attempted to establish similar lines for the unspliced β-globin, but its expression levels in induced cells were too low to enable robust quantification. The tiles were selected based on the change in their localization specifically in the spliced and not in the unspliced context  tail, which are absent from circular RNAs, which may have to recruit such factors indirectly. Another aspect to consider is that circular RNAs can adopt more compact folds compared to linear forms of the same sequence, as was recently demonstrated54, which can influence the ability of RBPs to access and bind specific sequences, and thus influence RNA export.MPRNAs were recently used for the identification of RNA elements driving nuclear enrichment in different settingsIn this study, we used backbones based on three sequences—the β-globin, circPVT1, and a scrambled sequence of circPVT1. As our focus was on effects of sequences on localization rather than on circularization, in the circPVT1 and SCRcircPVT1 backbones we inserted the tiles into the center of the circRNA, minimizing the effects of the variable sequence on circularization. We further used an exon-junction-spanning primer for library generation, which allowed us to ignore sequence changes that would affect localization and/or stability by affecting back-splicing efficiency. We note that the high correlation between circPVT1 and the SCRcircPVT1 backbones, and the lack of similarity to the linear version of circPVT1, all suggest that the backbone sequence has an overall limited effect on the activity of sequence elements that influence localization and/or stability.9, whereas regulation of circRNA subcellular localization or stability have not been extensively studied. The post-transcriptional fate of circRNAs is particularly interesting, as the main known pathways for export and decay of long RNA heavily rely on the 5′ cap and the poly(A) tail, which are absent in circRNAs. The paucity of these access points for decapping and deadenylation are thought to underpin the relative stability of circRNAs, and are likely a key feature in the functionality of some circRNAs, as they can allow circRNAs to remain stable while bound to RBPs that typically trigger decay though exonucleolytic activities. Here we report that cytoplasmic localization and stability of circRNAs are also correlated with each other, albeit weakly, supporting the notion that long-lived RNAs accumulate in the cytoplasm, which they likely reach when the nucleus disassembles during mitosis. Nevertheless, the variability we observe in both stabilities and Nuc/Cyto ratios of different circRNAs suggest that specific pathways, such as SRSF1 binding, sculpt the specific post-transcriptional fate of individual circRNAs.While it is now well appreciated that a large variety of circRNAs are produced in mammalian cells, their biology remains poorly understood. Much emphasis has been put on studying factors contributing to circRNA biogenesis and their potential translation56, and that exonic splicing enhancers are associated with efficient NXF1-dependent export25, although in those studies SR proteins had a positive effect on export, whereas for SRSF1 we observe a negative effect. Depletion of SRSF1/SF2 ortholog in D. melanogaster, was shown to lead to accumulation of various circRNAs in fly cells57. It has been suggested that SRSF1 primarily inhibits circRNA biogenesis, but our results suggest that it also inhibits circRNA export, which may also limit circRNA accumulation. Indeed, SRSF1 KD resulted in increased expression of several circRNAs , and was shown to associate with its canonical target, β-actin mRNA, in the nucleus63, consistent with a broader role in RNA export. There is also evidence that it may contribute to the export of short non-coding RNAs64. IGF2BP proteins were previously shown to have highly overlapping target sites65, and their combined or independent depletion led to a mild reduction in expression of their targets in HeLa and HepG2 cells66, while IGF2BP1 depletion did not preferentially affect levels of the bound targets in human embryonic stem cells49. Despite the fact that these factors bind closely similar motifs, their KD has a divergent effect on the fate of their targets in MCF-7 cells. IGF2BP1 contributes to RNA export and to its destabilization in the cytoplasm, as its KD leads to both nuclear retention and an increase in the expression of the targets. In contrast to IGF2BP1, IGF2BP2 KD led to a reduction in target expression levels, in both the nuclear and the cytoplasmic fractions. In both cases, the effects are strongest for spliced RNAs, and no strong effects are observed in a circular context.IGF2BP (IMP/ZBP) factors have been extensively studied in the context of RNA stability and localization62. These limited effects could stem from the limited potency of individual short sequences that typically act in the context of much longer RNAs, and it is unclear how individual short sequences synergize together in the context of long RNAs. For example, the sequences we identified as influential when using the β-globin backbone may synergize in specific ways with the known elements in β-globin that inhibit export28. It is also possible that imperfections in the nuclear/cytoplasmic fractionation procedure, where some residual cytoplasmic RNA occasionally remains associated with the nuclei, limit the ability to detect large effect sizes. Notably, a series of recent MPRNAs applied to neurite/soma fractionations, some using the same experimental design as we do here, occasionally found much larger effect sizes, even though in many cases no short elements could be found to recapitulate the activities of full-length 3′UTRs69.One limitation of our study is that the sizes of the effects on Nuc/Cyto ratios elicited by the individual tiles are typically quite modest and rarely exceed 2-fold. Such effects are comparable to those observed in other studies using a similar system70. The Wilusz laboratory has shown that circRNA size is important for their export pathway and efficiency16. In our setting, we studied circRNAs of a fixed length of 560–590 nt, corresponding to the ‘intermediate’ length range in ref. 16, where circRNA export can be regulated by either UAP56 or URH49. We have previously found that RNA length is also associated with the export efficiency of linear RNAs71, and showed that the export of unspliced or long-exon-bearing RNAs is NXF1-dependent and mediated by structured and G/C-rich regions25. Relatedly, Kudla and colleagues have observed a strong dependence on G/C-content of the export of unspliced but not spliced RNAs72, findings which also have support in studies of individual genes73. Future studies will elucidate how transcription efficacy and chromatin status, splicing, polyadenylation, RNA size, RNA modifications, and G/C context, all synergize with specific sequence elements recruiting RBPs to sculpt RNA fate.Beyond the RNA form, we expect that additional features will have important roles in influencing how a specific short sequence affects RNA fate. The transcriptional context of the host RNA, and specifically whether the elements are transcribed from chromosomes or from episomal vectors can play a substantial role, due to the tight coupling between transcription and export2 incubator at 37 °C. Cells were routinely tested for mycoplasma contamination using Mycoplasma test kit .MCF-7 (ATCC) were maintained in DMEM (Gibco) medium, supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Pen-Strep , in a humidified 5% CO22 and synthesized by Twist Bioscience (San Francisco). Backbone sequences and insertion positions are given in Supplementary Data Oligonucleotide pools were designed as in ref. 26. In brief, 10 µg of RNA from nuclear and cytoplasmic RNA was incubated with RNase R  for 30 min at 37 °C. Treated RNA was isolated using miRNeasy Mini Kit (Qiangen) following the manufacturer’s instructions and eluted with 30 µl of nuclease-free water. RNA was then incubated with polyA polymerase  for 30 min at 37 °C. 100 µl of Oligo-d(T)25 Magnetic Beads  were washed one time with lysis/binding buffer according to manufacturer’s instructions and were suspended in X2 lysis/binding buffer. RNA was incubated at 70 °C for 5 min, and was then incubated with the washed beads. Samples were incubated with shaking for 20 min. RNA samples with Oligo-dT beads were placed on a magnetic stand and the supernatant was collected. RNA was isolated using miRNeasy Mini Kit. Libraries for sequencing were generated using SENSE Total RNA-Seq Library Prep Kit .RPAD was performed according to the protocol as described in ref. 75. Chimeric junctions within 5 nt of the annotated circRNA boundaries of each annotated circRNA were counted and used to compute Cyto/Nuc ratios.RNA-seq reads were mapped to the genome with STAR with the following parameters: “–outSAMstrandField intronMotif –outSAMtype BAM SortedByCoordinate –outWigType bedGraph –chimOutType SeparateSAMold –chimSegmentMin 15 –alignSJoverhangMin 15 –alignSJDBoverhangMin 15 –chimScoreSeparation 10 –chimJunctionOverhangMin 5”. The chimeric read pairs identified by STAR were parsed and compared with circRNA boundaries annotated in CircBase and in publications focused on circRNAs in breast cancer cells and in other cell types76; Oligonucleotide pool was amplified by PCR using Q5 High-Fidelity DNA Polymerase , and purified using AMpure beads  at a 2:1 beads:sample ratio according to the manufacturer’s protocol. For adding flanking regions of target plasmid, PCR product was amplified using primers containing parts of the β-globin, circPVT1, or SCRcircPVT1 constructs (Supplementary Data Cloning of CircLibA and NucLibA into expression vectors was performed using restriction-free (RF) cloning77 and intersected with ENCODE eCLIP clusters as in ref. 22.RNA-seq libraries from nuclear and cytoplasmic fractions of HepG2 cells from the ENCODE project were quantified using human RefSeq annotations using RSEM6 cells were seeded in a 10 cm plate (Corning). Plasmid transfections were performed using PolyEthylene Imine (PEI) 78.Twenty-four hours prior to transfection, 102, 10 mM EDTA, 1 mM DTT, 0.5% NP-40, 10 U/ml RNase inhibitor), and incubated on ice for 5 min. The extract was centrifuged for 5 min at 300 × g in a cold centrifuge, and the supernatant was transferred to a new tube and centrifuged again for 1 min at 500 × g. The supernatant (cytoplasmic fraction) was transferred to a new tube and RNA was extracted using TRIREAGENT. The nuclear pellet was washed once in 150 ml RLN buffer, resuspended in 1 ml of buffer S1 , layered over 3 ml of buffer S3 , and centrifuged for 10 min at 2800 × g in a cold centrifuge. The supernatant was removed and RNA was extracted from the nuclear pellet using TRIREAGENT.Cells were washed in cold PBS and detached from plates by 10 mM EDTA, a fraction was transferred to a new tube and RNA was extracted using TRIREAGENT  to obtain whole-cell extract (WCE). Remaining cells were washed in cold PBS, resuspended in 150 ml RLN buffer . cDNA was synthesized using 1 μg of RNA (measured by Nano-drop spectrophotometer), with qScript Flex cDNA kit (Quanta Biosciences). Quantitative real-time PCR (qRT-PCR) was performed using Fast SYBR qPCR mix (Thermo Fisher) and analyzed on ViiA7 real-time PCR machine (Applied Biosystems) in a final reaction volume of 10 μl. The primer sets used for qRT-PCR are listed in Supplementary Data 2O. Product was used for amplification with barcoded primers, and the amplified libraries were purified by two-sided AMpure purification; First with a 0.5:1 beads:sample ratio followed by a 0.7:1 ratio. Libraries were sequenced a NextSeq 500 or NovaSeq 6000 machine to obtain 150 nt single-end reads.One microgram of RNA was used for cDNA production using the qScript Flex cDNA synthesis kit  and a gene-specific primer containing library 3′ adapter and part of the Illumina RD2 region. The entire cDNA reaction was diluted into 100 mL second strand reaction with a primer containing a unique molecular identifier (UMI), library 5′ adapter and part of the Illumina RD1 region. The second strand reaction was carried for a single cycle using Phusion Hot Start Flex DNA Polymerase , purified using AMpure beads at a 1.2:1 beads:sample ratio and eluted in ddHMCF-7 cells were transfected with plasmid libraries as described above. Twenty-four hours post-transfection, growth medium was replaced and actinomycin D  was added to a final concentration of 5 µg/ml. DMSO  was used as control. Cells were collected after 2, 4, and 8 h of actinomycin D or DMSO treatment using TRIREAGENT spiked with 2 RNA oligos transcribed in vitro from a DNA template encoding Renilla Luciferase at concentrations of 0.002 ng/ml and 0.0002 ng/ml. Libraries were prepared as described above, and expression levels of the tiles were normalized to luciferase levels.Half-life was calculated by first fitting a linear regression model between the time points and the expression levels. The intercept and slope were extracted from the regression equation. The half-life was then calculated as (logb(0.5)-lm_intercept)/lm_slope.Templates for in vitro transcription were generated by amplifying 2 regions of Renilla Luciferase sequence from a plasmid using Phusion Hot Start Flex DNA Polymerase , with primers that contain the adapters of NucLibA and CircLibA  sequence preceding the adapter. Each read was then matched to the sequences in the library, without allowing insertions or deletions. The matching allowed mismatches only at positions with Illumina sequencing quality of at least 35 and we allowed up to two mismatches in the first 15 nt (‘seed’), and no more than four overall mismatches. If a read matched more than one library sequence, the sequence with the fewest mismatches was selected, and if the read matched more than one library sequence with the same number of mismatches, it was discarded. The output from this step was a table of counts of reads mapping to each library sequence. Only fragments with at least 20 reads were used in subsequent analysis. We then used these to compute nuclear/cytoplasmic and WCE/plasmid ratios after adding a pseudocount of 0.5. Tiles sequences, GC content, DeltaG, expression levels and Nuc/Cyto ratios in each context are listed in Supplementary Data The sequenced reads were used to count individual library tiles as in ref. 79) and GENCODE v26 annotations, and gene expression levels were quantified using RSEM77. To quantify subcellular localization, Nuc/Cyto ratios were calculated using RSEM output of genes with average FPKM > 1. Differential expression in total, Cyto and Nuc samples were computed using DESeq2  with default parameters. Genes parameters, expression levels in WCE, cytosol and nucleus and normalized Cyto/Nuc ratios are listed in Supplementary Data WCE, cytosolic and nuclear fractions obtained after KD of IGF2BP1 and IGF2BP2 and fractionation were used to generate libraries using the SENSE mRNA-Seq Library prep kit (Lexogen) according to manufacturer’s protocol and sequenced on a NovaSeq 6000 machine to obtain 100 nt paired-end reads. RNA-seq reads were mapped to the human genome (hg19 assembly) with STAR  cloningMCF-7 cells were infected by the addition of growth medium containing lentiviral particles and 8 μg/ml Polybren (Sigma). Selection was done by the addition of Puromycin (InvivoGen) at a concentration of 1 μg/ml. Pools of infected cells that survived the selection were used for smFISH; cells were transfected with siRNAs (see above), and 32 h after transfection Doxycycline  was added to a final concentration of 1 µg/ml. Cells were grown for an additional 16 h and were fixed using 4% PFA in PBS.52. Library targeting β-blogin consisted of 22 probes labeled with Quasar 570  software. Quantification of cytoplasmic and nuclear signals was performed with default parameters and recommended filters of FishQuant.Probe libraries were designed according to Stellaris guidelines and synthesized by Stellaris  as described in Raj et al.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses. Picornaviridae, Flaviviridae, Coronaviridae and other viral families (Coronaviridae family) has spread worldwide for the past two years, threatening lives by causing severe symptoms in patients and resulting in millions of deaths (Flaviviridae family) was estimated to infect 71 million people worldwide, and the number of infected people has increased by nearly 2 million a year, causing cirrhosis, hepatocellular carcinoma, liver failure and even death (Picornaviridae family) threats human health by its extensive outbreak and causing deaths  N protein to promote viral replication, and inhibition of hnRNP A1 could result in reduced virus copy numbers of different strains of PEDV in CCL81 cells  see Fi. InteresHnRNP A1 also affect the replication of viruses from other virus families , 53. HnRHnRNP A2 has been confirmed to interact with Japanese encephalitis virus (JEV) NS5 by binding to the 5’ UTR of the negative-stranded RNA to enhance viral replication . Additio3C2 tetramers in native hnRNP complexes  . The RC/Flaviviridae family is also reported to be regulated by hnRNP C1/C2  motif  have been identified and named based on their molecular weight: p37h) motif . Isoformsequence . All fousequence .AUF1 significantly promotes the replication of several members of the Flaviviridae family, including Zika virus (ZIKV), West Nile virus (WNV), DENV and HCV , and siRNA-mediated knockdown of hnRNP D remarkably downregulated viral replication  during high-dose infection with CVB3 and EV71 . Stress HnRNPs E1 and E2 are also known as poly(C)-binding protein-1 (PCBP-1) and PCBP-2. The remaining two members (hnRNP E3 and E4) are divergent from hnRNP E1 and E2 , 90. UnlHnRNP E were predominantly reported to be associated with stem-loops of viral RNA, within cloverleaf or IRES –97. DuriDuring EV71 infection, hnRNP E1 binds to stem-loops I and IV within the 5’-UTR after being recruited to the viral membrane-associated complex to facilitate replication . APOBEC3This KH domain-dependent hnRNP E-viral RNA interaction could protect PV RNA from 5’ exonuclease and maintain viral RNA stability of coxsackieviruses, echoviruses, and rhinoviruses . StabiliCalicivirus IRES was slightly distinct from type I IRES of PV, it constituted an important GNRA tetraloop in subdomain d10c. This tetraloop was identified to bind to the KH1 and KH2 domain of hnRNP E2, along with another subdomain d10b within IRES binding to the KH3 domain of hnRNP E2. And the KH2 domain of hnRNP E2 is required for efficient calicivirus replication initiation . HnRNP EHnRNP I, also known as PTBP, regulates splicing by binding to polypyrimidine stretches at a branch point upstream of exons. Similar to the structure of hnRNP L, hnRNP I composed of four RRMs. Both hnRNP L and hnRNP I participate in RNA-related biological processes, including mRNA stabilization, pre-mRNA and translation. Both PTBP1 and PTBP2 can interact with CU repeats to repress nonconserved cryptic exons .During positive single-stranded RNA virus infection, hnRNP I can regulate viral IRES-dependent translation and viral replication –117. As HnRNP K is a versatile RNA-/DNA-binding protein that is involved in multiple fundamental processes of gene expression and signalling, including chromatin remodelling, RNA splicing, mRNA stability, transcription and translation . FurtherDuring HCV infection, hnRNP K was identified to interact with the HCV core protein and 5’ UTR of HCV RNA , 128. ItHnRNP K was suggested to bind to the IRES of both EV71 and FMDV RNA, although the binding sites of hnRNP K on the IRES were slightly different , 132. StFor JUNV, hnRNP K was confirmed to be recruited from the nucleus to the cytoplasm during infection to favour virus propagation . SINV nsSimilar to other members of hnRNPs, hnRNP L is involved in mRNA stabilization, mRNA transportation, pre-mRNA splicing and IRES-mediated translation. HnRNP L was first identified as a member of the hnRNP family. In particular, it has been reported that hnRNP L-directed RNA switches regulate the stress-dependent translation of vascular endothelial growth factor A (VEGFA) and promote cell apoptosis . Furtherpol in the presence of FMDV RNA, it was speculated that hnRNP L inhibits viral RNA synthesis in the replication complex (HCV could recruit cellular eukaryotic initiation factors (eIFs) and ITAFs to the IRES elements, initiating and modulating translation through a complicated network of RNA–protein and protein–protein interactions and the contact between the 5’- and 3’-ends of the viral genome . HnRNP L complex . And for complex , 142, moAs a component of the spliceosome complex, hnRNP M (or CEAR) is abundant in the nucleus and comprises four different isoforms generated by alternative splicing. The four isoforms constitute three RRMs with shifting locations , 143. HnHnRNP M silencing can increase the replication of SINV, CHIKV and Semliki Forest virus (SFV), indicating hnRNP M could impede virus infection. And it is worth mentioning that hnRNP M, hnRNP C and hnRNP E1 colocalize with viral replicases in the cytoplasm . In contA subsequent study reported that loss of hnRNP M results in hyperinduction of a cohort of inflammatory and antimicrobial genes in VSV-infected macrophages, enhancing macrophage antiviral defences and controlling virus infection. This finding reveals that hnRNP M could restrain macrophage antiviral functions and positively regulate virus replication . During HnRNP M also associate with innate immune pathways to regulate virus infection , 153. WhUnlike other hnRNP proteins, hnRNP F and H appear to bind poly(G)-rich tracts, whose RRMs are not conserved and described as quasi-RRMs (qRRMs). In addition to regulating the maturation and posttranscriptional processing of pre-mRNA, hnRNP H/F protein was recently found to localize to stress granules in response to cellular stress. Although recognizing similar sequences, hnRNP F was upregulated, while hnRNP H and H2 were significantly down-regulated during Nipah virus infection . With 2DOther than interacting with positive single-stranded RNA viruses, hnRNPs could also affect life cycle of retrovirus . For exaDespite their diversity in structure (from hnRNP A1 to U), hnRNPs are involved in multiple cellular processes, including pre-mRNA processing, mRNA transport, regulation of translation, and controlling miRNAs , 165. Tovia interacting with viral RNA or proteins to aid their life cycle proceeding , the China Postdoctoral Science Foundation (2020M683651XB), the earmarked fund for China Agriculture Research System (CARS-42-17), China Agriculture Research System of MOF and MARA and the Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (SCCXTD-2020-18).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acyrthosiphon pisum virus (APV), in the host aphid’s adaptation to unfavorable plants. Based on the transcriptomes of the pea aphid Vicia faba colony and Vicia villosa colony, 46 single nucleotide polymorphism (SNP) sites were found in the APV genomes from the two aphid colonies. One SNP at site 5,990, G5990A, located at the RNA-dependent RNA polymerase (RdRp) domain, demonstrated a predominance from G to A when the host aphids were shifted from V. faba to the low-fitness plants V. villosa or Medicago sativa. This SNP resulted in a substitution from serine (S) to asparagine (N) at site 196 in RdRp. Although S196N was predicted to be located at a random coil far away from conserved functional motifs, the polymerase activity of the N196 type of RdRp was increased by 44.5% compared to that of the S196 type. The promoted enzymatic activity of RdRp was associated with a higher replication level of APV, which was beneficial for aphids as APV suppressed plant’s resistance reactions toward aphids. The findings showed a novel case in which mutations selected in a symbiotic virus may confer a favor on the host as the host adapts to new environmental conditions.Host adaptation has the potential to cause rapid genetic variation in symbiotic microorganisms in insects. How mutations in symbiotic viruses favor viral fitness in hosts and even influence host adaptability to new environments remains elusive. Here, we explored the role of genetic divergence at one site of a symbiotic virus,  After treatment with the TURBO DNA-free kit  to remove genomic DNA contamination, RNA was reverse-transcribed to cDNA using the SuperScript™ III first-strand synthesis system (Invitrogen) and Oligo-dT primers  in accordance with the manufacturers’ instructions.V. faba colony, V. villosa colony, and the V. villosa colony that was raised on V. faba for 1–5 months and 27 individual aphids from the M. sativa colony were extracted for cDNA synthesis. A 460 bp fragment encompassing site 5,990 was amplified using the primer pair G5990A-F/G5990A-R  cells for protein expression. After overnight induction with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 16°C, the cells were pelleted by centrifugation and resuspended in lysis buffer  for sonication in ice water. The supernatant was retained for the RdRp enzymatic assay.Two types of APV RdRp, S196, and N196, were subcloned into the pET28a vector to generate recombinant plasmids with His-tags using the primer pair RdRp-his-F/RdRp-his-R . The recCP containing a T7 RNA polymerase promoter at the 5′ terminus was amplified using cp101-T7F/cp101-R primers (in vitro transcribed to CP RNA as an RNA template with T7 RNA polymerase (Promega) according to the manufacturer’s instructions. The enzymatic assay was performed in a total volume of 25 μl containing 20 mM Tris–HCl (pH 8.0), 1.5 mM MnCl2, 2 mM MgCl2, 100 mM NaCl, 4 mM DTT, 10 U RNasin (Promega), 250 μM NTP , 0.5 μl of RNA template (800 ng/μl) and 100 μg of crude RdRp-His protein. The reaction mixture was incubated at 25°C for 2 h, and then 30 μg of proteinase K (Ambion) was added to terminate the reaction. The RNA products were extracted with phenol/chloroform (1:1), precipitated overnight using 95% ethanol containing 0.3 M sodium acetate and dissolved in 10 μl of RNase-free water.A 101 bp fragment of APV  primers  and in vvia dot blot. The RNA products were first spotted onto a BrightStar™-Plus positively charged nylon membrane  and then placed in a UV crosslinker  for 10 min. The biotin-labeled RNA products were detected with a Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific) according to the manufacturer’s instructions. The RdRp-His protein in the crude extracts was measured by western blotting using an anti-His monoclonal antibody . The RdRp enzyme activity was represented by the relative intensity of biotin-labeled RNA products to RdRp-His, which was quantified with ImageJ software. The 1 mM and 0.1 mM biotin-16-UTP were used as positive controls. One hundred micrograms of crude protein from the cells transformed with pET28a vector and 20 mM Tris–HCl (pH 8.0) buffer were used as negative controls. Six replicates were prepared. Differences were analyzed using Student’s t test with SPSS 17.0 software.The biotin-labeled RNA product was detected V. faba colony and V. villosa colony; the APV-free third-instar nymphs that were fed on an artificial diet containing the APV crude preparations from the V. faba colony or V. villosa colony for 1 d  to asparagine (N). In the APV genome from the M. truncatula colony, which was previously sequenced , which is also a picorna-like virus , APV RdRactivity . Motif Vactivity . The S oactivity . The thr similar , althoug similar .E. coli cells and used for enzymatic activity measurement with an RNA template and biotin-labeled NTP. The amounts of synthesized RNA products were compared between the two types of RdRp after normalization by the RdRp protein amounts. The results showed that the N196 type of RdRp produced more RNA products than the S196 type  and the National Natural Science Foundation of China (no. 32102207).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Trypanosomatids are protozoan parasites that cause devastating vector-borne human diseases. Gene expression regulation of these organisms depends on post-transcriptional control in responding to diverse environments while going through multiple developmental stages of their complex life cycles. In this scenario, non-coding RNAs (ncRNAs) are excellent candidates for a very efficient, quick, and economic strategy to regulate gene expression. The advent of high throughput RNA sequencing technologies show the presence and deregulation of small RNA fragments derived from canonical ncRNAs. This review seeks to depict the ncRNA landscape in trypanosomatids, focusing on the small RNA fragments derived from functional RNA molecules observed in RNA sequencing studies. Small RNA fragments derived from canonical ncRNAs  were identified in trypanosomatids. Some of these RNAs display changes in their levels associated with different environments and developmental stages, demanding further studies to determine their functional characterization and potential roles. Nevertheless, a comprehensive and detailed ncRNA annotation for most trypanosomatid genomes is still needed, allowing better and more extensive comparative and functional studies. Discobids, formerly part of the eukaryotic lineage of excavates, which are no longer supported as a monophyletic clade. Thus, these organisms comprise a very early divergent group in the eukaryotic evolutionary tree and show many distinctive features, which are not present in well-studied fungi, plant, or animal cells, providing numerous insights into the early establishment of cell and molecular mechanisms of eukaryotic . . T. cruzOverall, these observations indicate a unique tsRNA expression pattern favoring specific processing, sorting, or stabilization of tsRNAs in diverse conditions, compartments, or life stages of the parasites. Several reports show that different tsRNAs are usually synthesized/processed in normal parasite cell biology while under certain stress, which seems to be a common strategy to respond to the environmental changes. Despite tsRNAs being the most studied sncRNA fragments derived from canonical ncRNAs, further studies are needed to define the molecular mechanism and the precise relation to cell biology, stages, and pathology of the parasites. The small nuclear RNAs (snRNAs) are highly conserved U-rich non-coding RNAs located in the nucleus, which are essential in the spliceosome complex, the machinery involved in pre-mRNA processing . The majInterestingly, small RNA fragments derived from specific locations of the snRNAs were found in eukaryotic model organism ,63,64, aT. cruzi [T. brucei [L. donovani and L. braziliensis found snRNA-derived fragments and identified one snsRNA in the top 20 most abundant fragments in the libraries [In trypanosomes, small RNA-seq experiments showed fragments derived from snRNAs with a median of 40 nts. However, 82.1% of the fragments were mapped to snRNAs U4 and U5 [T. cruzi ,51,53 an. brucei ,36,55, bibraries .Small nucleolar RNAs (snoRNAs) are conserved ncRNAs with a length between 60 and 250 nts, encoded in introns of host genes or controlled by independent promoters. Their main function is to serve as a guide for specific base modifications in the ribosomal RNA. Briefly, the snoRNAs are classified into two groups depending on the modification they guide on rRNA: the box C/D snoRNAs serve as a guide for 2′-O-ribose methylation, and the box H/ACA snoRNAs serve as a guide for pseudo-uridylation . While tT. cruzi, a sncRNA sequencing experiment revealed the presence of sdRNAs with a median length of 35 nts derived from both classes of snoRNAs (C/D box and H/ACA box) [T. brucei (implemented for snoRNAs gene annotation) found small RNA fragments of 20–30 nts derived from snoRNAs. Small RNA fragments were identified for 80% of C/D box and 90% of H/ACA box snoRNAs; however, the authors speculate that derived fragments could appear as the result of low-level RNA degradation during preparation and/or manipulation [L. donovani and L. braziliensis identified a small proportion of sdRNA (~1.5%) [The discovery of stably accumulating fragments derived from snoRNAs (termed sdRNAs) led to new regulatory functions besides the known canonical main function of this molecule ,74,75,76ACA box) . Then, tpulation . Posteripulation ,51,53,55pulation . However (~1.5%) . OverallRibosomal RNAs (rRNA) are non-coding RNAs transcribed by RNA polymerase I that are indispensable components of a large RNP complex, the ribosome, essential for the translation of all proteins in the cell. Indeed, ribosomal RNA is the most abundant RNA in the cell, reaching at least 80% of the RNA mass of the cell . The smaT. cruzi revealed that 17% of reads derived from rRNA and showed a three-peak length distribution of 20, 33, and 46 nt [T. cruzi, 25% of reads mapped to rRNAs [T. brucei [T. cruzi epimastigotes revealed that most of the sncRNAs (95%) identified were fragments derived from rRNA [T. cruzi (epimastigotes and metacyclic trypomastigotes) determined that the most abundant class in all samples were the sdrRNAs, supporting previous observations [T. cruzi (carrying large amounts of sdrRNAs) provoked changes in the gene expression level of many genes in host HeLa cells upon its incorporation [T. cruzi trypomastigotes display a 5-fold increase in invasion over cells pre-incubated with trypomastigotes Evs [T. cruzi epimastigotes under nutritional stress revealed that sdrRNAs were the most abundant class of small RNAs, being 46% and 58%, respectively [T. brucei parasites exposed to different environmental conditions [L. donovani and L. braziliensis which revealed a 31% and 15% of reads mapping to rRNAs, respectively [L. donovani (39 nts) and L. braziliensis (52 nts). In contrast with previous observations, site-specific enrichments could not be determined for the fragments derived from rRNA genes, and 90% of the mappings were distributed in both 28S and 18S rRNA genes [T. brucei [In trypanosomes, a small RNA sequencing experiment of epimastigotes of nd 46 nt . Similarto rRNAs . Remarka. brucei . Further. brucei . Posterirom rRNA . Then, trvations ,53. Remaporation . Indeed,otes Evs . The comectively . Howeverectively . Additioectively . Recentlnditions . These fectively . RemarkaNA genes . It is w. brucei . The lat. brucei . These n. brucei   is broadly conserved, playing key roles in defense against invading viruses, regulation of gene expression, programmed DNA rearrangements, and genome surveillance ,88. The T. brucei have functional RNAi, which involves a canonical AGO, two Dicers (one nuclear and one cytoplasmic), and two so-called RNA Interference Factors [L. braziliensis, L. guyanensis, L. panamensis) have functional RNAi pathways comprising orthologues of AGO and Dicer genes [L. braziliensis are mainly derived from SLACS and TATE transposable elements (~75%) [L. braziliensis, and thus they might be involved in intercellular communication [T. brucei and L. braziliensis RNAi pathways show organism-specific diversifications with implications for the production of dsRNA and siRNA [In the Trypanosoma genus, only  Factors ,93,94,95 Factors ,94,96,97 Factors . Further Factors . Among Ler genes ,101. Thes (~75%) . Remarkanication . Overallnd siRNA . The sizL. major, L. donovani, L. mexicata and L. tarentolae) contains only remnant, highly degenerated AGO1 pseudogenes, or lack one or more of the RNAi pathway genes [Strikingly, the siRNA pathway became non-functional in the remaining trypanosomatids ,101. Theay genes ,101. Thray genes . It is way genes .Vault RNAs (vtRNAs) are a class of eukaryotic non-coding RNAs of generally 84 to 140 nt, transcribed by the RNA polymerase III, firstly identified for its association with the vault particle ,105,106.T. brucei [T. brucei, based on secondary structure prediction and sequence similarities of short stretches in the 5′ and 3′ regions that form the bulged terminal helix [L. braziliensis vtRNA of approximately 190 nt was validated and represents the longest known member of the vtRNA family in eukaryotes. Interestingly, it was demonstrated that the silencing of the vtRNA in the bloodstream form of T. brucei resulted in decreased production of the SL exon-enriched long RNA products  with miRNA-like functions but generated by a different pathway from canonical miRNAs biogenesis ,111,112.RNA editing was first observed in trypanosomes when the insertion of four non-DNA encoded uridine nucleotides was described to restore the coding capacity of the mitochondrial cytochrome C oxidase subunit 2 gene, which presents a frameshift in the genomic sequence . Later, Trypanosomes are distinguished by the presence of a single mitochondrion containing a kinetoplast nucleoid composed of a bipartite mitochondrial genome and histone-like basic proteins. The kinetoplast DNA (kDNA) consists of a densely packed network of interlinked circular DNA molecules of two kinds, referred to as maxicircles and minicircles. Maxicircles are equivalent to the mitochondrial genome in most organisms, thus encoding ribosomal RNAs, two ribosomal proteins, and 16 respiratory-related genes, mostly cryptogenes, meaning they need to be edited in order to restore a protein-coding capacity. This editing is abundant as there can be hundreds of editions for each mitochondrial mRNA. The edition is directed by hundreds of guide-RNAs mostly encoded by the multicopy (102–103) ~1 kb DNA molecules named minicircles . Editing guide RNAs (gRNAs) are small RNAs (50–60 nt) with a 5′ triphosphate and a 3′ oligo-U tail . T3/T7-lT. brucei [T. brucei. A different approach was carried out in L. Tarentolae [The nearly complete repertoire of gRNA molecules was determined by RNA sequencing for the procyclic stage of . brucei , describ. brucei , and it rentolae , using tT. brucei when a common 39-nucleotide sequence, named spliced leader (SL), was found at the 5′-end of different variant surface glycoprotein transcripts [T. brucei) with a short half-life. The SL-RNA is transcribed from multiple loci in the genome, being this gene is the only one under the control of conventional RNA polymerase II promoter sites in trypanosomes [Trans-splicing involves the precise joining of two distinct RNA molecules, and this spliceosomal processing was firstly described in nscripts ,127. Latnscripts , while tnscripts . Since tnscripts . The leaanosomes ,132. Up P. falciparum [Since the initial discovery of telomerase as the solution to the end-replication problem of eukaryotic chromosomes, much progress has been made in the identification of telomerase core components: the catalytic telomerase reverse transcriptase (TERT) and intrinsic template-bearing telomerase RNA (TR) . From thlciparum . T. brucei TR (TbTR) has several unique attributes that suggest a mechanistically different process of telomere maintenance [T. brucei, the TbTR of 900 nt RNA (longer than vertebrates TR) is encoded by a single-copy gene transcribed by pol II [Besides common features, ntenance . In T. by pol II ,136. Desy pol II ,134. In y pol II ,136,137.y pol II . Since ty pol II ,140. T. brucei [T. brucei genome. Only a few have been experimentally characterized with proposed roles in translation regulation [Long non-coding RNAs (lncRNAs) are ncRNAs longer than 200 nt that do not code for functional proteins. Several thousand of this RNA biotype are transcribed by pol II in eukaryotic genomes; as an example, the human genome may contain more than 100.000 lncRNA genes . Even th. brucei . However. brucei ,144, mosgulation  parasitegulation ,144. Intgulation   . HoweverSmall RNA fragments derived from several canonical ncRNAs  were identified as the most commonly studied trypanosomatids, displaying changes in their levels among developmental stages and diverse growing conditions, as well as their distribution over subcellular compartments. These observations demand further studies for characterization and functional analysis with special emphasis on defining possible mechanisms of action. The latter could expose new layers of post-transcriptional gene expression regulation, a crucial level of control in the biology of these parasites. Furthermore, considering that in trypanosomatids the miRNA pathway is absent, while the siRNA pathway is present only in some lineages of the phylogenetic tree, functional characterization may be challenging, but could offer interesting new insights into the mechanism of action.