The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal “UC” sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications. The authors present the structure of Cas13bt3 in an activated state, illustrating its target length-dependent activation mechanism, and use it as a guide to develop a high-fidelity Cas13bt3 variant suitable for RNA knockdown and therapeutic applications. Upon proper target RNA binding and pairing with the spacer segment of the crRNA, the Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) nuclease domains within Cas13 are activated to cleave the bound RNA in cis (on-target cleavage activity) and any surrounding non-specific single-strand RNA (ssRNA) in trans 3. Cas13 has been widely adapted for in vivo RNA knockdown and transcriptome modulation9, due to its high efficiency and programmability in RNA targeting. However, the in vivo applications of CRISPR-Cas13 systems are hindered by non-specific cleavage of the activated Cas13, which could damage the transcriptome and cause cell death12. In addition, the large size of Cas13 effectors (967–1152 amino acids) limits the possibility of packaging them into adeno-associated viruses and subsequent in vivo delivery for therapeutic applications14. A deep mechanistic understanding of the Cas13 nuclease activation and cleavage will be critical to modulate the Cas13 collateral activities and reduce the effector size for in vivo applications.The CRISPR-Cas13 system is an RNA-guided ribonuclease with a single Cas13 protein effector and a CRISPR RNA (crRNA)17. The structural basis of Cas13 HEPN nuclease activation has been investigated for several Cas13 systems24. In both Cas13a and Cas13d, target binding induces inter-domain conformational changes and brings the two catalytic HEPN domains closer to each other to allow cleavage of both the target and nonspecific RNAs21. Recently, a series of compact Cas13 proteins (~800 amino acids) suitable for in vivo applications have been reported26 and structures of the binary and ternary complexes of the compact Planctomycetes bacterium Cas13bt325  were determined27. Interestingly, the HEPN1 and HEPN2 domains of Cas13bt3 move in unison over 24 Å upon target binding. However, the two HEPN domains adopted the same conformation relative to each other in both structures27, and how this rigid body movement of the HEPN domains induces nuclease activation was unclear. Moreover, the ternary complex of Cas13bt3 was prepared with a short, 25 nucleotide (nt) target RNA27, which does not support optimal Cas13bt3 activation7. Further structural and mechanistic studies are essential to illustrate the molecular basis of Cas13bt3 activation and guide the engineering of Cas13bt3 for in vivo applications.Four subtypes of Cas13 (a–d) have originally been characterized, each having its own unique domain organization and crRNA sequence, exhibiting different levels of on-target and collateral cleavage activitiesHere, we illustrate the allosteric activation mechanism of Cas13bt3, which guides the rational engineering of a highly specific Cas13bt3 variant. Biochemically, Cas13bt3 cleaves target and nonspecific RNA at internal “UC” sites and displays target length-dependent activation. A structure of the activated Cas13bt3 in complex with crRNA and 30 nt target RNA was determined at 3.5 Å with a cryo-electron microscope (cryo-EM). Compared to previous structures, the HEPN nuclease domains exhibit almost 50 Å of target length-dependent translocation along the target RNA. Coupled with the movement of HEPN domains, two inter-domain linkers (IDLs) that bridge the HEPN domains and crRNA binding domains become fully stretched and form new interfaces critical for target RNA binding and Cas13bt3 activation. Based on the proposed allosteric mechanism, mutagenesis on or around the IDLs produced new Cas13bt3 variants with minimal collateral activity while maintaining target cleavage activities in biochemical assays and mammalian cells. We expect the similar mechanism and engineering strategies to be applicable to other Cas13 systems.25, we designed a crRNA with a 30 nt spacer for testing Cas13bt3 activities against target and nonspecific RNA. Cas13bt3 is inactive in the absence of a crRNA whereas crRNA binding activates Cas13bt3 to efficiently cleave a 122 nt target RNA (T0) , likely around a “UUC” sequence present in the ssRNA36  Fig. . InteresT0) Fig. , which i7, although 2-3 nt terminal mismatches can be well tolerated. Since a 25 bp spacer-target RNA duplex was used to assemble the complex and only a 20 bp duplex was observed in the previous ternary complex27, the previously reported structure of Cas13bt327 might only represent an intermediate state during activation (Cas13bt3Int), meaning additional conformational changes are required for Cas13bt3 to become fully active.Next, we measured the target length dependence of Cas13bt3 activation and cleavage. We first varied the target length from 30 nt (T1) to 27 (T2), 24 (T3), and 21 (T4) nt, which exhibited 67%, 42%, and 6% collateral cleavage activity relative to the activity of T1 Fig. . We thenTo understand the structural basis of Cas13bt3 activation, we attempted to capture Cas13bt3 in a fully activated state with the catalytically inactive Cas13bt3 , crRNA and a 30 nt target. However, the dCas13bt3-crRNA-target ternary complex is unstable during cryo-EM sample freezing and no clear particles with the right size can be found on the cryo-EM grid. To stabilize the ternary complex, we designed a hairpin substrate by connecting the crRNA and target RNA with a 6 nt (CUUCUU) linker to stabilize the ternary complex Fig. . The resAct, the true active state according to our biochemical assays,) appears as a bilobed architecture with recognition (REC) and nuclease (NUC) lobes on two ends of the spacer/target duplex RNA (dsRNA)  Fig. . A 36 ntNA) Fig. . The RECNA) Fig. a, 5. MorNA) Fig. . The NUCbt3 Fig. . Insteadbt3 Fig. .Act structure is compared to previous Cas13bt3-crRNA binary (Cas13bt3Bin) or Cas13bt3Int structures  are scattered and only present in three areas, the Helical1 domain, the IDL1, and the HEPN1/HEPN2 domains  in mammalian cells, we took advantage of a well-established dual-fluorescence approach based on the plasmid (Addgene #190033) which contains the previously engineered high-fidelity Cas13X (hfCas13X or hfCas13bt3)20 and Cas13d21, where the dsRNA binding site is pre-formed and only enlarged by interdomain rotations upon target binding  with an N-terminal His6-SUMO tag following a Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 2 (SEPN2) cleavage site. The WTCas13bt3 was constructed by a In-Fusion Snap Assembly method (TaKaRa), while other Cas13bt3 mutations were constructed using the WTCas13bt3 as the template. The expression plasmids for WT and mutated Cas13bt3 will be available upon request.AgeI and HindIII-double digested backbones of the spacer-modified hfCas13bt3 plasmid. All the constructions were confirmed by sanger sequencing (Genewiz). The DNA primers for cloning were listed in Supplementary Table To test the collateral effects of Ca13bt3 mutants in mammalian cells, we generated the plasmids based on the template of hfCas13bt3 (Addgene #190033). First, the spacer sequence of guide RNA 1 (GTCCTCCTTGAAGTCGATGCCCTTCAGCTC), 2 (AGCACTG CACGCCGTAGGTCAGGGTGGTCA), or 3 (GCAGGACCATGTGATCGCGCTTCTCGTTGG) was generated by ligating phosphorylated and annealed oligos (IDT) into the corresponding BbsI-digested backbone of hfCas13bt3, respectively. Then, the dCas13bt3, and mutant containing Y643A or Y647A were constructed by the In-Fusion Snap Assembly method based on the plasmid of Cas13bt3.1 (Addgene #171379), and the gene sequence of WTCas13bt3, dCas13bt3, and mutant containing Y643A or Y647A was respectively ligated to 25. Briefly, recombinant Cas13bt3 protein was overexpressed in Escherichia coli Rosetta (DE3) in Terrific Broth (TB) medium. The cells were grown at 37 °C until OD600 reached 0.8, then the cells were cooled on ice for 20 min and switched to 18 °C and induced with 0.2 mM isopropyl b-D-1-thiogalac- topyranoside (IPTG) for 16–18 h. Cell pellets were harvested with centrifugation at 5000 × g at 4 °C, and resuspended in buffer A , and lysed by sonication, then centrifuged at 23,000 × g for 1 h at 4 °C. The supernatant was passed through 2 ml Nickel-NTA resin (Qiagen), and the resin was washed with 20 column volumes of lysis buffer, then the protein was eluted from the column with buffer B . The SUMO tag was cleaved overnight on ice by SEPN2 protease (homemade). After cleavage, the protein was loaded onto a 5 ml heparin column  which was preequilibrated with the buffer C . Protein was eluted by a gradient NaCl concentration with buffer D . Peak fractions were pooled, concentrated, and supplemented with 30% glycerol. The purified Cas13bt3 protein was aliquoted and flash-frozen at −80 °C for further study. The mutants were purified by the same method as described above.Expression and purification of Cas13bt3 were performed as previously described with modificationsHindIII and XbaI restriction sites. The construction was confirmed by sanger sequencing (Genewiz), then the transcription template was obtained by PCR amplification with primers of M13F and hpIVT-R. The PCR product was purified with phenol and followed by 95% ethanol precipitation, then the product was dried and dissolved with RNase-free H2O. The transcription templates of crRNA (crIVT) and short target RNAs (T1IVT-T23IVT) were ordered as oligonucleotides from IDT, and the double-stranded transcription template for each RNA was obtained by annealing the forward primer and the reverse primer at a molar ratio of 1:1. The constructions containg transcription template of long target RNAs  were prepared by the same method as hpRNA, and each transcription template was obtained by PCR amplification with M13F and T0-R or TUC-R, respectively. The DNA oligos for preparing RNA transcription templates and corresponding RNA sequences were listed in Supplementary Table The 120 bp transcription template for hpRNA containing 17 bp T7 RNA polymerase promoter and 103 bp transcription template was ordered as 8 DNA oligos . The S1 to S4 were phosphorylated with T4 polynucleotide kinase , annealed and ligated into the pUC19 vector (Thermo Fisher) with 2 for crRNA, hpRNA and long target RNAs or 8 mM MgCl2 for short target RNAs, 2 mM spermidine, 2.5 mM each NTP, 100 ng/μl DNA template and 2 μM T7 RNA polymerase (homemade). The RNAs were purified by 12% denaturing (7 M urea) polyacrylamide gel electrophoresis (PAGE), extracted, and precipitated by ethanol. The RNAs pellet was dissolved with RNase-free H2O, and the concentration were measured with Nanodrop (DeNovix DS-11). The crRNAs and hpRNA were diluted to 10 μM and annealed by heating to 75 °C for 5 min, then slowly cooling to room temperature, then aliquoted and flash-frozen at −80 °C for further use.The in vitro transcription was carried out at 37 °C for 4 h in buffer containing 100 mM HEPES potassium (pH7.5), 30 mM DTT, with 20 mm MgCl32P] ATP , and 1 μl of T4 polynucleotide kinase . The reaction was incubated at 37 °C for 30 min. The labeled product was purified with Illustra MicroSpin G-50 columns. For 5ʹ-end radiolabeling of ssRNA (R1-R17), the RNA oligos were ordered from IDT without phosphorylated 5ʹ end and were directly subjected to radiolabeling by the same method as described above. The concentration of 5ʹ-end radiolabeled RNAs was measured with Nanodrop. The eluted RNA was aliquoted and stored at −20 °C if not immediately used. The 5ʹ fluorophore (FAM) labeled target RNA T1 (5ʹ-FAM-T1), U20 (5ʹ-FAM-U20), ssRNA R1 (5ʹ-FAM-R1), and ssRNA36 (5ʹ-FAM-ssRNA36) or 5ʹ-fluorophore and 3ʹ -quencher labeled R1 (R1-FQ) were ordered from IDT.For 5ʹ-end radiolabeling of target RNAs, the phosphorylated 5ʹ end of target RNAs from IVT was removed by phosphatase (NEB) and purified with Illustra MicroSpin G-50 columns , then 2 μM of de-phosphorylated target RNA was incubated in a 20 μl reaction mixture containing 1 X T4 polynucleotide kinase buffer, 0.5 μl of [γ-2, and 1 mM DTT. The complex was purified through two steps of gel filtration chromatography, first at high salt to purify the Cas13bt3 complex with specific hpRNA binding and second at low salt to reduce the salt concentration for stabilizing the ionic interactions between Cas13bt3 and hpRNA . The peak fractions of the complex were collected, concentrated, and the buffer was exchanged to the buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, and 1 mM DTT with an Amicon Ultra centrifugal filter unit with 50 kDa cutoff (Millipore). Second, the above 500 μl concentrated complex was further purified with Superdex 200 Increase 10/300 column in buffer . The peak fractions of the complex were collected and concentrated to 0.9 mg/ml, as determined by the Bradford assay kit (Bio-Rad).The complex of Cas13bt3-hpRNA was assembled by mixing the purified dCas13bt3 and the 103 nt hpRNA at a molar ratio of 1:2 in buffer containing 20 mM HEPES, pH 7.5, 400 mM NaCl, 5 mM MgCl3 μl freshly purified Cas13bt3_hpRNA complex were spotted onto freshly glow-discharged Quantifoil R 1.2/1.3 Cu 300 mesh grids (Quantifoil) or GF 1.2/1.3 AU 300 mesh grids (Protochips). Excess samples were blotted using the Vitrobot Mark IV (FEI) with the standard Vitrobot filter paper (Ø55/20 mm (Ted Pella), the blotting time was set to 2 s, the blotting force was set to 5 and the blotting was done under 100% humidity at 20 °C. The grids were flash-frozen in liquid ethane and stored in liquid nitrogen.32, and defocus values were estimated on non-dose-weighted micrographs with Gctf33, respectively.The images were recorded on a Titan Krios electron microscope operated at 300 kV  using the super-resolution mode with a nominal magnification of 165 K . Movies were recorded with a K2 Summit camera, with a dose rate of 7 e-/pixel/s with a total exposure time of 7 s, with 0.2 s for each frame, generating 35 frames per micrograph. Defocus values range was set between 0.6 and 2.2 μM. For dataset 1, 2236 micrographs were collected from Quantifoil R 1.2/1.3 Cu 300 mesh grids. For dataset 2, 3358 micrographs were collected from GF 1.2/1.3 AU 300 mesh grids. Micrographs from datasets 1 and 2 were motion corrected by MotionCor235. The particles were extracted to a pixel size of 5.2 Å and were subjected to 2D classification, and good classes were selected and used as templates for reference-base picking of both datasets 1 and 2. Around 4,798,775 particles from dataset 1 and 7,511,757 particles from dataset 2 were picked. The picked particles were extracted to a pixel size of 5.2 Å and around 1,735,702 particles from dataset 1 and 918385 particles from dataset 2 were selected after 2D classification, respectively. The selected particles were extracted (0.87 Å/pixel), combined, and imported to cryoSPARC-3.336 for another round 2D classification, and about 1,760,928 particles were selected. The selected particles were separated into six classes by cryoSPARC ab-initial reconstruction, setting the maximum resolution to 8 Å, and one class with intact and clear protein and RNA features was selected for subsequent processing. To solve the preferred orientation issue, iterative ab-initial reconstructions were performed three more times by setting the maximum reconstruction resolution to 6 Å and separating into three classes each time, during each round of 3D classification only the class with clear protein and RNA features was selected. About 37228 particles were selected from cryoSPARC and were imported and subjected to CTF refinement, and Bayesian polishing in RELION-4.0. The particles after polishing were imported to cryoSPARC-3.3, and final refinements were done with the non-uniform refinement37, which yielded a map for subsequent modeling with global resolutions of 3.52 Å according to the Fourier shell correlation (FSC) = 0.143 criterion38. The local resolution was estimated by cryoSPARC-3.3. Additional processing details are summarized in Supplementary Fig. About 110 K particles were picked from 100 manually screened micrographs from dataset 1 based on reference-free auto-picking (Laplacian-of-Gaussian picking) through RELION-4.0https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb). The model of DR was adapted from PbuCas13b-crRNA binary structure (6DTD)23, and the model of double-stranded RNA formed by the spacer and target RNA was generated from Coot-0.9.6 modeling tools39. Each domain of Cas13bt3, and RNAs were manually docked into the cryo-EM density maps in Chimera40. The model was further manually rebuilt in COOT based on electron density, and refined in Phenix with real-space refinement and secondary structure and geometry restraints41. No clear electron density was observed for 6 nucleotides linker between the spacer and target RNA, poor electron density was observed for the residues 227–229 of the Helical1 domain and 272–278 of the Lid domain. All figures were generated by UCSF Chimera and PyMol (http://www.pymol.org). Statistics of all cryo-EM data collection and structure refinement are shown in Supplementary Table A homolog model of Cas13bt3 was generated with the AlphaFold Colab server  were incubated at a molar ratio of 1:1:1 on ice for 10 min in the cleavage buffer, then the reactions were initiated by mixing 50 nM or indicated concentration of Cas13bt3-crRNA-target complex (activated Cas13bt3) with 100 nM indicated reporter RNA. All reactions were performed at 37 °C for 30 min, and terminated by adding 2× loading buffer  and quenched at 85 °C for 5 min. Quenched reactions were resolved on 12% denaturing polyacrylamide gels (Urea-PAGE) for target RNA cleavage assays, or 20% Urea-PAGE for collateral RNA cleavage assays. For the assays using 5′-end radiolabeled RNAs as substrates, the gels were followed by exposure to a phosphorimager plate for 1 h, and imaged using phosphorimaging by the Sapphire Biomolecular Imager (Azure Biosystems). For the assays using 5′-FAM-labeled RNAs as substrates, the gels were directly visualized and captured with a Sapphire Biomolecular Imager (Azure Biosystems). Assays were performed in at least three independent replicates, and the band intensity of the substrates and products were analyzed using Azure Spot (Azure Biosystem) and plotted through GraphPad Prism.Both target RNA cleavage and collateral cleavage reactions were performed in a cleavage buffer containing 20 mM Tris.HCl, pH 8.0, 60 mM NaCl, 2 mM MgClFor Supplementary Fig. For Supplementary Fig. For testing the collateral activity of Cas13bt3 mutants, 1 μM of WTCas13bt3 or mutants and crRNA were incubated at a molar ratio of 1:1 on ice for 10 min in the cleavage buffer first, then 10-μl cleavage reactions were initiated by mixing 50 nM protein-crRNA complex with 100 nM target RNA T1, and 100 nM R1-FQ in cleavage buffer. All reactions were performed in a 384-well microplate  at 37 °C, with fluorescence monitored  every 5 min for 60 min on a TECAN infinity M200 plate reader, and data were normalized to the first-time point.2,1 mM DTT, 5% glycerol. WTCas13bt3 and mutants were respectively complexed with crRNA in binding buffer at a molar ratio of 1:1 for 15 min on the ice, then a serial dilution of protein-crRNA complex (from 50 to 300 nM) was incubated with 10 nM 5ʹ-FAM-T1 in binding buffer for 30 min on ice, respectively. Then each sample was mixed with a 5× loading buffer containing 10% glycerol and 0.05% bromophenol blue before loading onto a 5% native polyacrylamide gel containing 0.5× TBE buffer. The gel was pre-run at 4 °C for 45 min at 120 V with 0.5× TBE as a running buffer. Gels were imaged using phosphorimaging by the Sapphire Biomolecular Imager (Azure Biosystems). The bound and unbound fraction of target RNA was quantified by using Azure Spot (Azure Biosystem), plotted in GraphPad Prism, and fitted by specific binding with Hill slope.To quantify the target RNA binding affinity, assays were carried out in a binding buffer containing 20 mM Tris.HCl, pH 8.0, 60 mM NaCl, 2 mM MgCl2.HEK293T cells  were cultured in 10 cm treated, vented plates (Greiner Bio-One). The culturing media was Dulbecco’s modified Eagle’s Medium (DMEM) plus GlutaMAX (Gibco) which contained fetal bovine serum at 10% v/v (Gibco) and Penicillin-Streptomycin 1% v/v (Gibco). Cells were passaged between 80-90% confluency. The cell status used in all experiments was between Passage 1 to Passage 10. They were grown in an incubator set at 37 °C and 5% CO5 cells in 500 μl of DMEM, with cell vitality above 95% and plate confluency around 90%. Cell vitality was assayed through Typhan Blue (Invitrogen) staining, placed on a Countess Cell Counting Chamber Slide (Invitrogen) and counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific), with the average of the two concentration and vitality measurements used. After seeding, cells were left at room temperature for 15 min to mitigate evaporation.To prepare for an experiment, cells were seeded at a concentration of 2 × 10After 12 h, cells were transfected. For each well, 800 ng of plasmids were buffered to a final volume of 25 μl in Opti-MEM I Reduced Serum Medium (OMEM) (Thermo Fisher Scientific). Along with the DNA, 2.5 μg of Polyethyleneimine (PEI) (Thermo Fisher Scientific) was buffered to a final volume of 25 μl in OMEM. The two were mixed and allowed to incubate at room temperature for 10 min before the total 50 μl was transfected into each well.Forty-eight hours later, cells were analyzed for mCherry fluorescence and EGFP fluorescence by flow cytometry using a SONY SA3800. Single stain controls were predetermined for all experiments by using HEK293T cells transfected with either a single mCherry reporter or a single EGFP reporter. All flow cytometry data were analyzed using FlowJo v10.4.0.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3’UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1’s RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG’s abundant RNA antagonizes TbRAP1’s silencing effect, thereby sustaining its full-level expression. VSG expression is essential for Trypanosoma brucei survival. Competition between TbRAP1’s RNA and dsDNA binding activities ensures that TbRAP1 sustains a high level expression of the active VSG while silencing other VSGs globally.Monoallelic  Many genes that undergo MAE encode cell surface receptors. For example, each human and mouse olfactory sensory neuron expresses only one odorant receptor gene1. Several microbial pathogens, including Trypanosoma brucei, employ MAE as part of their antigenic variation strategy5.Monoallelic gene expression (MAE), or allelic exclusion, is essential for many organisms. Notable examples include genome imprinting, X chromosome inactivation, and random monoallelic expression of autosomal genes in mammalsTrypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. It sequentially expresses distinct Variant Surface Glycoproteins (VSGs), its major surface antigen, to evade the host’s immune surveillance. VSG is monoallelically transcribed by RNA polymerase I6 from one of the ~15 nearly identical VSG expression sites (ESs)8. In these subtelomeric polycistronic transcription units, VSG is always the last gene located within 2 kb of the telomeric repeats8. Parasites that express multiple VSGs are more rapidly eliminated by the host9, underscoring the importance of VSG MAE for T. brucei survival. Many factors affect VSG MAE, such as nuclear lamina, the inositol phosphate pathway3, transcription elongation11, and a subtelomere and VSG-associated VEX complex14.TbRAP1, a nuclear and essential telomere protein, is a key regulator of VSG MAE18. Depletion of TbRAP1 leads to derepression of silent VSGs up to a thousand-fold18. The TbRAP1-mediated silencing is stronger at loci closer to the telomere than those further away15. We recently reported that TbRAP1 possesses a dsDNA binding activity mediated by its R/K patch (737RKRRR741) in the DNA binding (DB) domain (aa 734–761)18. This dsDNA binding activity is essential for TbRAP1’s association with the telomere chromatin and TbRAP1-mediated VSG silencing. However, we do not understand the underlying mechanism of how TbRAP1 selectively permits the active VSG to be fully expressed while silencing other VSGs.Our previous studies have demonstrated that TbRAP1, while known RAP1 homologs have not been reported to have any RRM domains. Assisted by the R/K patch, TbRAP1 RRM binds the 16-mer consensus sequence of VSG 3’UTRs20 in vitro, while TbRAP1 binds the active VSG RNA in vivo. Strikingly, mutations in the RRM domain that specifically abolish the TbRAP1-VSG RNA interaction lead to an acute decrease in the active VSG RNA level by ~50% and subsequent derepression of all silent VSGs, thus disrupting both aspects of VSG MAE. In contrast, mutations in the R/K patch alone or in both the R/K patch and RRM lead to acute depression of silent VSGs, but the active VSG RNA is only moderately decreased by ~13%. Mechanistically, TbRAP1’s RNA and dsDNA binding activities compete in a substrate concentration-dependent manner. Such competition suggests a mechanism of VSG MAE where the active VSG’s abundant RNA antagonizes TbRAP1’s dsDNA binding-mediated silencing effect at the active VSG locus to sustain its full-level expression.Here our NMR studies identify an RNA Recognition Motif (RRM) in 21. None of the known RAP1 homologs has been reported to have any RNA binding activity. We previously found that TbRAP1 does not bind the telomeric repeat-containing RNA (TERRA) in RNA IP experiments24. However, to our great surprise, we found that TbRAP1 interacts with the active VSG RNA  tag17. Quantitative RT-PCR (qRT-PCR) analysis detected significantly more active VSG2 RNA in the TbRAP1 CLIP product than in the negative control IgG CLIP product , small nuclear RNA gene activation protein 50 (SNAP50), and Protein Kinase A catalytic subunit (PKAC1) were also examined in CLIP products. Approximately the same amount of TbTERT, SNAP50, and PKAC1 RNAs were detected in both TbRAP1 and IgG CLIP products  is responsible for binding to the active VSG RNA, we first determined the solution structure of TbRAP1639-761 by NMR spectroscopy 26 with the characteristic topology of a four-stranded anti-parallel β-sheet and two α-helices packed behind the β-sheet  A1 bound with an RNA oligo [10.2210/pdb5MPG/pdb]28 2, an oligo that contains the TERRA sequence24. (UUAGGG)2 did not induce any noticeable chemical shifts when titrated to TbRAP1639-733  or GST-tagged TbRAP1 fragments were used , TbRAP1639-7332FL (F655LF694L), or TbRAP1639-7332FA (F655AF694A) did not  by Cre, as RRM mutants can interact with WT TbRAP1 through its BRCT domain17. Removal of the TbRAP1 F allele was confirmed by PCR 18 were expressed at a subtly lower level than WT TbRAP1  to test whether the RRM domain is necessary for tin Fig. , presumatin Fig. . ImmunofAP1 Fig. . Hence, TbRAP1F/∆RRM, TbRAP1F/2FQ, TbRAP1F/2FL, TbRAP1F/2FA, and TbRAP1F/2FA&5A after a 30–48 h Cre induction  in the active VSG2 RNA level, while RNA levels of silent VSGs increased several hundred-fold  and TbRAP1F/2FA&5A cells (~87% by 36 h after Cre induction)  Fig. . Our temVSGs at the time points of 12–36 h after Cre induction in TbRAP1F/mut cells. Notably, derepression of silent VSG 3, 6, and 9 at 12 h after the Cre induction in RRM point mutants was only ~10 fold, significantly milder than the ~100 fold observed in TbRAP1F/5A cells   were both incubated with TbRAP1639-761, no ternary complex of TbRAP1-RNA-DNA was observed   or 170-VSG-UTR by TbRAP1639-761 are comparable in the range of ~100–300 nM  Fig. 18. Howevved Fig. . InsteadUTR Fig. . SimilarGG) Fig. . EMSA es nM Fig. , thus alDNA Fig.  as expecDNA Fig. . TherefoTbRAP1 MybLike folds into an RRM module with a canonical βαββαβ topology, in addition to a C-terminal flexible loop corresponding to the DB domain18. Similar to other RRM domains, the four-stranded β-sheet in TbRAP1 RRM is a functional RNA binding site, with conserved residues F655 and F694 poised to form stacking interactions with RNA substrates27. Hence, TbRAP1 is different from known RAP1 homologs, which do not contain RRM or possess any RNA binding activity. Thus, our study uncovers an additional important function of this essential telomere protein.Our NMR studies reveal that TbRAP1 RRM by NMR titration, fluorescent polarization, and EMSA in vitro, which is also confirmed by RNA CLIP in vivo. In vitro, TbRAP1 RRM alone can bind the 16-mer consensus sequence of VSG 3’UTRs20. This binding explains why TbRAP1 interacts with all types of active VSG RNA in vivo. Moreover, RRM domains are known to be promiscuous and capable of recognizing many RNA sequence motifs. As a result, it is probable that TbRAP1 may bind to RNA sequences aside from the consensus 16-mer of VSG 3’UTRs. Indeed, our NMR titration data shows that TbRAP1 RRM bound to 35-VSG-UTR with stronger affinity than 16-VSG-UTR, and TbRAP1 RRM also binds a 170-nt long RNA without any VSG2 3’UTR sequence in the EMSA experiment. In addition, it is possible that TbRAP1 RRM may recognize the structural features of VSG 3’UTRs, as they have been predicted to form a common secondary structure20. Future experiments are needed to determine additional RNA sequences, both within and outside VSG RNAs, that are recognized by TbRAP1 RRM.We have validated the RNA binding activity of TbRAP1 DB enhances the RRM-mediated RNA binding activity. This effect is particularly significant for recognizing short RNA oligos in vitro, such as 16-VSG-UTR. As the DB domain is a long and flexible loop with little inter-domain interaction with RRM, it is possible that RRM and DB contact different parts of the RNA substrate independently. This bi-valent binding mode may achieve higher binding affinity than RRM alone. This combinatorial effect to enhance RNA binding affinity has been reported in other RNA binding proteins. In particular, many RNA splicing proteins such as FUS and hnRNPU contain intrinsically disordered arginine-rich RS or RGG repeats adjacent to well-folded RNA-binding domains such as RRM or Zinc Finger (ZF) domains34. These disordered motifs are capable of sequence-independent RNA interaction and have been reported to synergize with RRM or ZF to enhance overall RNA binding35. Additionally, the dual roles of the DB domain in mediating both TbRAP1’s DNA and RNA binding offers a mechanistic underpinning for mutually exclusive and concentration-dependent competition between the two activities.Interestingly, we found that VSG RNA levels reveals an essential role of TbRAP1 RRM in maintaining the full-level expression of the active VSG, which is a critical aspect of VSG MAE. We observed a striking decrease of ~50% in the active VSG RNA level in RRM point mutants. For two reasons, this decrease most likely did not result from a reduced amount of available RNA polymerase I for transcribing the active VSG, even though silent VSGs were derepressed globally. Firstly, TbRAP1 DB mutants such as ∆DB and 5A only affect the active VSG RNA level subtly, even though silent VSGs were derepressed up to ~1000 fold. Secondly, at 12 h post Cre-induction, the active VSG RNA level in RRM mutants is already significantly lower than that in the 5A mutant, while silent VSGs have not been derepressed to the same extent as that in the 5A mutant. Furthermore, the decreased active VSG RNA level in RRM point mutants is not caused by RNA processing, as the half-life of the active VSG RNA is not affected by RRM mutations, and TbRAP1 is primarily a nuclear protein as shown in IF analyses , treated by DNase (Qiagen), and purified using the RNeasy column (Qiagen). cDNA was synthesized using a random hexamer and the MMLV reverse transcriptase (Promega) according to the manufacturer’s manual. cDNA and γH2A ChIP product were analyzed by real-time PCR on a CFX Connect (Bio-Rad) using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad) according to the manufacturer’s manual. rRNA level was measured and used as a loading control. Data acquired on CFX Connect were processed using MS Excel and Graphpad Prism. qPCR primer sequences are listed in Supplementary Table For qRT-PCR, total RNA was isolated from TbTRF rabbit antibody30, or IgG conjugated with Dynabeads-Protein G (ThermoFisher) for three hs at 4 °C. In γH2A ChIP, the total lysate was equally divided into two fractions, each incubating with 1 µg of γH2A rabbit antibody23 or IgG conjugated with Dynabeads-Protein G. After washing, IPed products were eluted from the beads and DNA was isolated from the products followed by Southern slot blot hybridization or quantitative PCR analysis.200 million cells were cross-linked by 1% formaldehyde for 20 min at RT with constant mixing, and the cross-linking was stopped by 0.1 M Glycine. Chromatin was sonicated by a BioRuptor for 6 cycles (each 30 sec sonication and 30 sec rest) at the high level to get DNA fragments of ~500 bp on average. After saving a small amount of sonicated sample as the input fraction, the sample was equally divided into three fractions, each incubating with 1 µg of HA monoclonal antibody 12CA5 (MSKCC Antibody & Bioresource Core), All recombinant proteins used in this study are listed in Supplementary Table E. coli strains for optimum expression   as nitrogen source and D-Glucose  as carbon source. Protein expression was induced by IPTG. TrxA-His6 (TH6)-tagged proteins were purified with His•bind resin (Millipore) or NiNTA agarose (Qiagen) according to the manufacturer’s protocol. GST-tagged proteins were purified with Glutathione Sepharose 4 Fastflow beads (GE) according to manufacturer’s protocol. Purified proteins were dialyzed in dialysis buffer  at 4 °C overnight. Affinity purified TH6-tagged proteins were dialyzed in 3C protease reaction buffer . A total of 4 mg of dialyzed protein was digested with 140 units of Pierce™ HRV 3C Protease (ThermoFisher Scientific) at 4 °C overnight with nutation. The digestion mix was passed through NiNTA agarose column (Qiagen) to remove TH6 and 3C-His6. Tagless protein was collected from the flow-through fraction and concentrated using a Centricon (Millipore).Recombinant protein expression constructs were transformed into various Kd) between 16-VSG-UTR and each of the recombinant TbRAP1639-761, TbRAP1639-7615A, and TbRAP1639-733 proteins, serial ten two-fold dilutions of the proteins were prepared in the binding buffer . The highest protein concentration started at 1200 μM, and 5’ 6-carboxyfluorescein (6-FAM) labeled 16-VSG-UTR (Sigma-Aldrich) was diluted to 50 nM. 40 µl protein-RNA mixtures were incubated for 20 min at room temperature. FP signals were detected at 25 °C using a CLARIOstar  multi-mode microplate reader equipped with polarization filters with excitation wavelengths at 482 nm (482–16 mode) and emission wavelengths at 530 nm (530–40 mode), respectively. The anisotropy value was obtained with a unit of millipolarization (mP). Focus and gain were adjusted by a reference well containing FAM-labeled RNA only. The Kd value was analyzed by fitting a nonlinear regression curve with one site-specific binding mode in GraphPad Prism.Fluorescence polarization (FP) experiments were conducted in black 384-well microplates with triplicates. To determine the equilibrium dissociation constant  at room temperature for 30 mins. Samples were electrophoresed in a 0.8% agarose  according to the manufacturer’s protocol. Radiolabeled RNA was gel-purified by 10% denaturing PAGE. Purified RNA was resuspended in 40 µl of RNase-free ddH2O. 35-32P alpha dCTP in a 50 μl of reaction  at room temperature for 60 mins. The radiolabeled probe was purified by 3 ml Sephadex G-50 column and precipitated overnight in 0.2 M sodium acetate pH 5.5/Ethanol followed by washes with 70% Ethanol and resuspension in 50 μl of ddH2O.A total of 150 ng of double-stranded linear DNA was radiolabeled using the Klenow fragment (NEB) and Kd value was analyzed by fitting a nonlinear regression curve with one site-specific binding mode in GraphPad Prism. TbRAP1 fragments without any tag was used in EMSA for calculating Kd. The protein purity is >90% . Titration curves were generated by plotting protein concentration vs percentage shift of the radiolabeled probe. The TbRAP1639-761 were 0.1 mM for the 15N-HSQC spectra, 0.6 mM for 2D 1H-1H-NOESY and 1.0 mM for 15N-NOESY, HNCACB, CACB(CO)NH, 13C-HSQC and 13C-NOESY experiments. NMR samples were prepared in 100 mM phosphate buffer  with 90% H2O/10% D2O or 99.9% D2O. NMR spectra were acquired on Varian Inova 500, 750, or 800 MHz spectrometers at 298 K. The data were processed using NMRPipe42 and analyzed using Sparky43 and CCPN44. Backbone and side-chain resonance assignment were achieved via the standard heteronuclear triple resonance correlation experiments using 15N, 13C-double labeled TbRAP1639-761. Interproton distance restraints were generated from 2D/3D NOESY experiments using a mixing time of 100 ms. Hydrogen bond restraints were generated base on the nuclear overhauser enhancement (NOE) patterns and derived from Talos45. Initial structure models were generated using CNSsolve46 using interproton distance, dihedral angle, and hydrogen bond restraints. Final structure refinement was performed using Xplor-NIH 3.347 using an implicit solvent potential48. Ten best structures of TbRAP1639-761 without restraint violation were selected.The concentrations of 15N-HSQC spectra were acquired with 0.1 mM 15N-labeled protein samples in 20 mM sodium sulfate, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 6.5. NMR titrations were performed by adding unlabeled concentrated RNA (1-5 mM) to 15N labeled protein (0.1 mM) gradually. NMR spectra were acquired on Varian Inova 800 MHz spectrometer at 293 K.4, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose) were UV crosslinked (800 mJ) in a UV Stratalinker 2400 (Stratagene). Cells were then harvested by centrifugation, resuspended in the IP buffer , 40 units RNaseIn, 1% TritonX-100, 0.1% SDS, 100 µM TLCK, 1 µM Pepstatin A), and incubated on ice for 30 mins. Samples were centrifuged at 15,800 x g and 4 °C for 15 mins and the supernatant is collected as the lysate. 10% of the lysate was saved as input. The rest lysate was equally divided into two fractions, each incubating with 1.2 µg of monoclonal HA antibody 12CA5 (MSKCC Antibody & Bioresource Core)/TbRAP1 rabbit antibody18 or IgG conjugated with Dynabeads Protein-G (ThermoFisher) at 4 °C for 3 h with rotation. The IPed products were washed with wash buffer  three times followed by washing with 1 X PBS once. After washing, the IP products were treated with proteinase K (200 µg) for 30 mins at 50 °C shaking at 450 rpm in a Thermomixer. RNA was then isolated from the IPed products using RNA STAT-60  followed by DNase treatment and RNA purification over an RNeasy column (Qiagen). Reverse transcription was done using MMLV (Promega) according to the manufacturer’s protocol followed by quantitative PCR using primers specific to various genes  were treated with 10 µg/ml Actinomycin D (Sigma) for 0, 15, 30, 45, 90, 120, or 150 mins. 40 million cells were harvested at each time point for isolation of total RNA using RNA STAT-60 . RNA samples were treated with DNase and purified on an RNeasy column (Qiagen). Quantitative RT-PCR was done the same way as described above. Data were processed using MS Excel and Graphpad Prism.TbTRF, TbRAP1, VSG6 rabbit antibodies and VSG3 monoclonal antibody were diluted 1:1 K; TbTRF chicken antibody was diluted 1:200) at RT for 2 h and the secondary antibody at RT for 1 h. Cells were then washed with 1 X PBS/0.2% cold fish gelatin/0.5% BSA and 1 X PBS followed by staining with 0.5 µg/ml DAPI before mounting coverslips on slides. Images were taken by a DeltaVision Elite deconvolution microscope. Images were deconvolved using SoftWoRx.Cells were fixed with 2% formaldehyde at RT for 10 min, permeabilized in 0.2% NP-40/1 X PBS at RT for 8 min, blocked by 1 X PBS/0.2% cold fish gelatin/0.5% BSA at RT twice, each for 10 min, followed by incubation with the primary antibody  on ice for 15 min. After washing 3 times with growth medium, cells were incubated with MACS beads conjugated with a rat anti-mouse IgM antibody (Miltenyi) on ice for 15 min followed by washing with growth medium twice. The mixture was then loaded onto an LD column, and cells in the flow-through fraction were collected and plated on 96-well dishes. 1/6 of the collected cells  were evenly distributed onto three 96-well dishes. Similarly, 1/3  and 1/2  of the collected cells were evenly distributed into six and eight 96-well dishes, respectively. All recovered colonies were tested again by western slot blot using a VSG2 rabbit antibody , and VSG2-positive clones were excluded from switchers. Raw switching frequency was calculated by dividing the number of true switcher colonies by the initial cell number. To determine plating efficiency, cells were plated at 1 cell/well concentration onto 3 X 96-well plates. Plating efficiency was calculated by dividing the number of colonies grown up by 288. The final switching rate was calculated by normalizing raw switching frequency with plating efficiency and divided by the number of population doublings. Data were processed using MS Excel and Graphpad Prism.TbRAP1F/2FQ and TbRAP1F/2FA&5A cells for 30 h before total RNA was isolated and purified through RNeasy columns (Qiagen). All RNA samples were run on a BioAnalyzer 2100 (Agilent Technologies) using the Agilent RNA 6000 nano kit to verify the RNA quality before sent to Novogene for library preparation and RNA high throughput sequencing, which was the same as described in18.The Cre expression was induced by adding doxycycline in RNAseq data were analyzed by Novogene:Raw reads of fastq format were first processed through Novogene perl scripts. In this step, clean reads were obtained by removing reads containing adapters, reads containing poly-N and low-quality reads. At the same time, Q20, Q30 and GC content of the clean reads were calculated. All downstream analyses were based on the clean reads with high quality.T. brucei lister 427 genome TriTrypDB-45_TbruceiLister427_2018_Genome.fasta and its annotation TriTrypDB-45_TbruceiLister427_2018.gff were downloaded from the TriTryp DB and used as reference. Index of the reference genome was built using hisat2 2.1.0 and paired-end clean reads was aligned to the reference genome using HISAT2.The HTSeq v0.6.1 was used to count the read numbers mapped to each gene. FPKM of each gene was calculated based on the length of the gene and the reads count mapped to this gene.P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.Differential expression analysis of two conditions/groups  was performed using the DESeq R package (1.18.0). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


ABC transporters are molecular machines which power the solute transport using ATP hydrolysis. The structural biology of ABC transporters has been exploding for the last few years, and this study explores timelines and trends for various attributes such as structural tools, resolution, fold, sources, and group leaders. This study also evidences the significance of mammalian expression systems, advancements in structural biology tools, and the developing interest of group leaders across the world in the remarkably progressing field. The field started in 2002 and bloomed in 2016, and COVID years were really productive to the field. Specifically, the study explores 337 structures of 58 unique ABC transporters deposited in the PDB database from which P-glycoprotein has the largest number of structures. Approximately, 62% of total structures are determined at the resolution of 3-4 Å and 53% of structures belong to fold IV type. With progressive advancements in the field, the field is shifting from prokaryotic to eukaryotic sources and X-ray crystallography to cryoelectron microscopy. In the nutshell, this study uniquely provides the detailed snapshot of the field of structural biology of ABC transporters with real-time data. ATP-binding cassette (ABC) transporters are members of a large superfamily of transporters which bind and hydrolyze ATP to move a diverse library of substrates across the membrane –3. ABC tAdvancements in structural tools such as X-ray crystallography and cryoelectron microscopy (cryo-EM) in combination with improvements in mammalian expression systems have revolutionized the field of structural biology of ABC transporters and progressed our understanding towards their mechanism , 10. TheEscherichia coli maltose transporter, which transports maltose or maltodextrins from periplasmic space to the cytoplasmic side [12 , heme, molybdate, and metal chelates, from outside to inside of bacterial cells. However, TMDs in fold II transporters consists of 10 transmembrane helices [2+, Co2+ and tryptophan across the cell membrane [2FG and MacB, respectively.Seven categories of structural folds have been defined in the structural biology of ABC transporters , 12  database in the last decade intrigued us to comprehend the field of structural biology of ABC transporters. The course of discovery of these structures is studied during time period spanning last 20 years, i.e., from the beginning (2002) of determination of the first structure of an ABC transporter to now (May 2022) , 18. Thihttps://rcsb.org), which enables open access to the accumulating knowledge of 3D structure, function, and evolution of biological macromolecules, was used to gather information regarding structures of ABC transporters [The workflow of the study is depicted in Figure sporters , 20. The2FG, TmrAB, WzmWzt, and BmrA) were determined using both structural tools   as well as eukaryotic (Saccharomyces cerevisiae) sources. As the usage of eukaryotic sources has been continuously increasing since 2012, the proportion of structures from prokaryotic sources (56%) is comparable to eukaryotic sources (44%)  from the beginning of the field to the present is 3.0-3.9 Å Figures . For exa% from thEscherichia coli, Homo sapiens, and Mus musculus whose proportions are 18%, 24%, and 10%, respectively (Figure Data collected on a number of structures determined from unique sources (Figure y Figure  with advy Figure , Table 3y Figure .As the expression systems and structural tools were advancing and revolutionizing the field, an accruing interest of new incoming group leaders in the field and their contribution should also be acknowledged Figure . The lasExploding field of the structural biology of ABC transporters with a number of deposited structures in the PDB database is significantly impacting the field by expanding our understanding towards structure-function relationships and informing critical aspects of mechanism in these transporters . As mentThis article provides an overview of the revolutionizing field of structural biology of ABC transporters. The data regarding structures of ABC transporters was collected from the PDB database and various trends with respect to sources, resolution, fold, structural tools, and group leaders were studied to understand the continuously growing nature of the field. In the nutshell, this study exclusively prepares the platform to build a focused database on structurally studied ABC transporters. It evidences the emergence of different structural folds during these years and their distribution among prokaryotes and eukaryotes. The study recognizes contributions of advancements in cryo-EM and mammalian expression systems and accruing interest of emerging group leaders in the field. Follow-up study on meta-analysis of studies on the utility of these structures in other scientific areas will be useful to understand the impact of the structural biology of ABC transporters. Furthermore, the structural alignment of these structures from different sources can deduce the relationships between structure, function, and evolution in this family.


The central role of RNA molecules in cell biology has been an expanding subject of study since the proposal of the “RNA world” hypothesis 60 years ago ,2. The iTwo review papers included in this Special Issue are resources for analysis and methods to study the biological roles of RNA molecules. The manuscript by Mathlin et al. discusses the current knowledge of epitranscriptomic marks and proposes a categorization schema based on the reference ribonucleotide and its rounds of modifications (“stages”) until reaching the given modified form . This coAnother review paper by Marasca et al. analyzes an oft-neglected topic in the field of RNA regulatory networks: the function of transposable genomic elements and their roles in cell physiology . TranspoMitochondrial RNAs are also discussed in this Special Issue. The article by Magistrati et al. focuses on how nucleotide modifications in mitochondrial rRNAs and tRNAs can be causative factors for mitochondrial diseases, referred to as modopathies, and how mutations in RNA-modifying enzymes can impair the efficiency of mitochondrial protein biosynthesis .Among the various families of small ncRNAs, microRNAs (miRNAs) are the most studied group since their discovery in model organisms. These promiscuous negative post-transcriptional regulators play central roles in cell and developmental biology and human disease. Two of the review papers included in this Special Issue describe the role of miRNAs in the regulation of proteostasis and protein aggregation during brain and skeletal muscle aging  and how Another article describes an alternative explanation of the effect exerted by myostatin in the control of muscle growth in mammals . Using mRNAs can also be dynamically interchanged between cells. Long-distance regulatory signals mediated by RNAs are often linked with their transport by vesicular conveyors. The RNA regulatory language is especially interesting when analyzing interspecies communication within holobionts involved in symbiotic or parasitic relationships, as discussed in the review paper by Leitão et al. .The plasticity of RNA function is achieved by a smart combination of structure, sequence, and nucleotide modifications. The structural features of RNA molecules are clearly related to their functions and can be used by cells to modulate biological functions, scaffold macromolecular complexes, and localize RNAs to specific cellular compartments. Two papers included in the Special Issue describe the role of G-quadruplex structures in the function of coding and non-coding RNAs. Niu et al. show experimental evidence of the presence of a G-quadruplex structure in the internal ribosome entry site (IRES) of the VEGFA gene transcript . In a ceThe functional plasticity of ncRNAs is also exemplified by two experimental papers showing the function of lncRNAs in different biological contexts. Fefilova et al. showed that the murine lncRNA Morrbid is a key player in the regulation of the splicing of the proto-oncogene NRAS  in hepatocytes . The altThe interface between RNA-binding proteins (RBPs) and specific RNA molecules is central to the formation of RNA–protein complexes and condensates . The rolMethods for the laboratory analysis of RNAs and their rigorous quality control are essential for the establishment of a solid knowledge base and are especially important in clinical applications. The article by Zhelankin et al. discusses the influence of pre-analytical factors in the profiling of circulating miRNAs . Using sThis Special Issue is concluded by two experimental papers describing how DNA methylation can be a source of variability in the expression of rRNA genes  and how Finally, we would like to acknowledge the serious, technically sound, and solid work presented by all of the authors who contributed to this Special Issue, together with the timely and constructive comments provided by all of the anonymous reviewers. We are in debt to you all, and we sincerely hope that we have the opportunity to collaborate with you again as we continue to explore the central role of RNA in cell biology, developmental biology, brain function, and transgenerational inheritance.


The mammalian suprachiasmatic nucleus (SCN) is the principal circadian clock that synchronizes daily behavioral and physiological responses in response to environmental cues. Here, we present a protocol for harvesting mouse SCN by vibrating microtome for diurnal transcriptome analysis. We describe steps for mouse entrainment, isolation of the SCN, tissue preparation, slicing with a vibratome, and handling of the harvested SCN for RNA extraction. This protocol can also be used for harvesting other mammalian brain regions for genomic studies.  •Extraction of highly viable SCN tissue from the adult mouse brain using vibratome•Precisely timed tissue harvest to detect circadian transcriptional rhythms•Full description of mouse entrainment, SCN collection, and rhythmicity analysis•A reference protocol for harvesting mammalian brain regions for genomic studies Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The mammalian suprachiasmatic nucleus (SCN) is the principal circadian clock that synchronizes daily behavioral and physiological responses in response to environmental cues. Here, we present a protocol for harvesting mouse SCN by vibrating microtome for diurnal transcriptome analysis. We describe steps for mouse entrainment, isolation of the SCN, tissue preparation, slicing with a vibratome, and handling of the harvested SCN for RNA extraction. This protocol can also be used for harvesting other mammalian brain regions for genomic studies. The protocol below describes the specific steps for harvesting and extracting RNA from the SCN. However, this protocol can be applied to any brain region of interest when followed judiciously.All animal studies were performed under the guidance issued by Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and Home Office Project Licence 19/0004. C57BL/6J were maintained and provided in-house by MRC Harwell.Timing: 2 weeks1.a.Place 8–10 cages in a specialized light-controlled circadian chamberb.Use a staggered light schedule when using multiple light-controlled circadian chambers for the ease of harvesting tissue(s) at distinct times during the day.Singly house adult mice (8–12 weeks old) in individually ventilated cages fitted with running wheels, under 12:12 h (hr) light-dark (LD) conditions (∼150 lux) with food and water available ad libitum.Note: For collecting tissues at night times, animals can be placed in cages inside a LCC programmed for lights on at 6 pm (ZT0) and lights off at 6am (ZT12). In this instance, for example, tissue(s) can be harvested from animals entrained at 8 am corresponding to ZT14.2.Entrain mice to the 12:12 h light-dark schedule for 8–10 days. Change cage bedding on day seven if required.CRITICAL: While the animals are under a light-dark schedule, minimize noise and cage disturbance as much as possible while maintaining animal welfare. It should be noted that light during lights-off would have a particularly detrimental effect on entrainment/experiment.The animals are entrained under 12:12 h (hr) light-dark conditions. The number of mice to be used in the study should be carefully planned. For daily transcriptome profiling, we recommend harvesting mouse tissue(s) in 4-h interval over 24-h period. We used 3–4 biological replicates per time-point, where each biological replicate was a pool of four individual tissues (SCN). In total, we entrained and used ca. 96 animals, composed of equal number of males and females for this study.Timing: 1 h3.a.https://actimetrics.com/products/clocklab/)Use ClockLab Analysis 6 software  of the entrained mice for last 7–10 days . HoweverNote: While the data and protocol outlined here utilizes ClockLab software to analyze wheel running activity, any in-cage activity monitoring system which assesses activity in relation to the timing of the light cycle can be used. Mice are nocturnal animals and show maximum activity in the dark phase; ZT12-ZT24.4.Discard animals that do not show robust rhythmic activity from the study.CRITICAL: Use consistent analysis settings such as percentile/normalized actogram while assessing the circadian behavior using ClockLab.•0.1% DEPC treated water: Add 1 mL of DEPC to 1 L of ddH20. Shake well to disperse the DEPC through the H2O under the fume hood. Incubate at 22°C–25°C for at least 12 h and autoclave at 15 psi on liquid cycle for 20 min to inactivate the remaining DEPC. Store at 22°C–25°C.•1X PBS (RNAse free): Add 100 mL PBS (10X) to 900mL DEPC treated water. Store at 22°C–25°C.•3% Agarose gel: Dissolve 3 g in 100 mL PBS  solution. Allow it to set for 20 min at 22°C–25°C. Store at 4°C for 30 days.Note: The solutions are stable at 22°C–25°C, and there are no particular time constraints regarding their preparation or use.CRITICAL: Diethyl pyrocarbonate (DEPC) is harmful with acute toxicity, Oral (Category 4), H302. Work under the fume hood and wash skin thoroughly after handling. Do not eat, drink or smoke when using this product.Timing: 2 days1.Following confirmation of stable entrainment, establish the correct dissection times for the time points needed.2.a.Wipe down dissection area and dissection tools with RNAseZap.b.Cut aluminum foil into small squares (approximately 4 cm by 4 cm), one square for each animal. Place one foil square onto dry ice.Prior to dissection time, set up dissection area.3.At the designated dissection time remove mouse from the cage, cull and dissect brain following institutional guidelines.Note: It is important to collect the tissue at a particular time-of-the-day even if it is not used for circadian studies as the transcript abundance greatly varies with time.4.a.If regions of the brain other than the SCN are to be used in subsequent analysis, the orientation of the brain on the foil may need to be changed so that the region of interest is not in direct contact with the foil. See ‘Place brain, cortex down, upon the foil square on dry ice.5.Allow the brain to freeze for 5 min. During this time, further dissections can be performed. .Figure 36.Once frozen, store the brain at −80°C until ready to continue to SCN dissection as specified below.CRITICAL: For dark-phase time points, perform the brain dissection under dim red light (no more than 50 lux). Desired intensity of the red light is maintained using filters blocking wavelengths below 600 nm. Avoid any exposure of light whilst collecting and freezing the brains.The brains are harvested from the entrained mice at designated time-points after cervical dislocation. Approximately 16 mice comprising of four biological replicates  are used per time-point.Timing: 2 weeks7.a.Place brain matrix, sterile razor blades, forceps, dissection needles etc. on dry ice to chill.Carry the stored mouse brains from −80°C freezer to the desired dissection area using a dry ice tray.8.Wipe the bench area, vibratome, light-dissection microscope etc. with RNaseZap to avoid contamination or degradation from RNAses.CRITICAL: Wear gloves throughout the procedure and periodically change or wipe with RNAseZap.9.https://campdeninstruments.com/products/7000smz-2-vibrotome) for coronal sectioning by following the specified guidelines in the equipment set up. Switch on the machine with the light source and scroll to the desired user and press [MENU]. The sectioning parameters associated with the user will display on the screen.a.Add ice to the tissue bath cooling jacket of the vibratome.b.Fill the metal tray with chilled 1% RNAse free PBS solution. See ‘c.Cover the vibratome metal chuck (removable tissue support) with tape. Use super glue to stick 1 × 1 cm block of 3% agarose gel at the curved edge of the chuck. This provides support for the mouse brain during gradual sectioning.Prepare the vibratome  on the brain matrix and cut approximately 3 mm from the caudal end by razor blade to remove the cerebellum.Note: Ensure the tissue is perpendicular to the brain matrix and the cutting line is in parallel with the grooves in the brain matrix. In doing so, it keeps the left and right hemispheres aligned on the same coronal section in order to minimalize angular variation in the dissection plane. This is especially important for non-circular and small nuclei.11.https://lafayetteinstrument.com/downloads/manuals/7000-v3.0%20Eng.pdf.Apply a tiny drop of superglue on the metal chuck. Now, gently transfer the brain from the matrix to the metal chuck with the rostral end facing upwards and affix the chuck on the metal tray as specified in the instruction manual CRITICAL: The metal chuck should be submerged in the RNAse free PBS, covering the top of the brain specimen.12.Manually set the bath to the start height following the manufacturer instructions.13.Bring the cutting blade to a suitable position by setting the start position using [ADVANCE] and the rotary knob.14.a.After a slice is cut, press [RETURN] to slide the blade back to the start position A.b.Section the mouse brain from rostral to caudal up to approximately 1.2 mm of Bregma.Press [SLICE ON/OFF] to begin and stop the sectioning.Note: Press [SLICE ON/OFF] to stop the sectioning once the blade reaches the top of the specimen, near the agarose block. After pressing [RETURN], if the slice is still attached to the specimen, dislodge it in the buffer gently with the help of a paint brush.15.Gently transfer the mouse brain slice(s) with the help of a paintbrush onto the glass slide placed over a cold block.16.Examine the mouse brain slice(s) collected between Bregma −0.3 mm and −0.8 mm for the presence of intact bilateral SCN  using th17.Select the appropriate brain slice and tease apart the SCN from the surrounding hypothalamic tissue using pre-chilled dissection needles and forceps .Figure 518.Aliquot 10 μL of Buffer RLT  on to the sub-dissected SCN and transfer into 1 mL RNAse free microfuge tubes (kept on ice) using 10 μL pipette tips.19.Place the tube on dry ice.Pause point: The dissected SCN can be stored in Buffer RLT for long term at −80°C.The dissected brains are placed on dry ice and sliced in the coronal plane into 250 μM thick sections using a vibrating microtome. The brain slice between Bregma −0.3 mm and −0.8 mm,Timing: 3 days20.Thaw the SCN tissues containing 1.5 mL tubes on ice for RNA extraction. Pool 3 individual SCN tissue samples suspended in Buffer RLT, collected at the same ZT in one separate RNAse free tube. This is referred as one biological replicate (BR) and labeled .CRITICAL: The tissue samples should be thawed completely on ice before transferring to another tube. Special care should be taken while pooling the samples to avoid cross-contamination.21.https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-kits) for purification of total RNA from microdissected cryosections using laser microdissection systems. The extracted RNA is finally eluted in 14 μl elution buffer supplied with the kit. During the RNA extraction, perform on-column DNase digestion as per manufacturer’s guidelines.Adjust the sample volume to 350 μl with Buffer RLT and follow steps for RNA extraction using an RNeasy micro kit (Qiagen) as per the manufacturer’s instructions  any suitable commercial RNA extraction kit such as RNAqueous Kit , PureLink RNA Mini kit  or TRIzol based RNA purification methodPause point: The extracted RNA can be stored for long term at −80°C.22.Inspect the quality and quantity of the extracted RNA for each sample on Bioanalyzer using Agilent RNA 6000 pico chip. For library preparation, do not include any RNA sample with RIN (RNA integrity number) < 7.0 . See ‘trNote: Alternatively, RNA gel electrophoresis can be used to check for the integrity of the RNA, and Nanodrop to assess the purity and the concentration of the extracted RNA. Ideally, 260/280 ratio of ∼2.0 from Nanodrop is accepted as “pure” for RNA.23.https://uk.neb.com/products/e7760-nebnext-ultra-ii-directional-rna-library-prep-kit-for-illumina#Product%20Information) and subsequently sequence the polyA enriched libraries on Illumina based platform (single or paired end).Prepare libraries for sequencing, in accordance with the manufacturer’s guidelines for a NEBNext Ultra II Directional RNA Library Prep Kit  eliminates the risk of accumulating uneven or broken sections and offers great advantage over cryostat sectioning when operated manually or at pre-programmed settings. In principle, this method of tissue dissection can easily be adopted to isolate any brain region of interest using cell density contrast. Further processing of the sub-dissected SCN tissues in RNAse free conditions will yield approximately 100 ng high quality RNA (RIN > 8.0) at a concentration of Timing: 2 dayshttps://usegalaxy.eu/) and identify transcripts with daily rhythmicity in the SCN using the R package dryR .https://www.r-project.org/.1.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (cut-off Phred < 20) and remove Illumina adapters by TrimGalore (v0.4.3)  .Assess the paired-end FASTQ files with FastQC (2.https://maayanlab.cloud/biojupies/) to generate raw count data.Align the quality checked and trimmed reads to mm10 genome assembly using STAR3.The gene counts generated in the above step are used to assess rhythmicity by R package dryR .4.https://github.com/naef-lab/dryR . In this instance, gene counts from four biological replicates per time-point  are used. Execute dryR specifying the condition(s) and time-series as instructed in the package guidelines. See ‘Prepare the data matrix file from the raw count data e.g.,  to serve5.The output file will be .csv file specifying the corresponding amplitude (amp), phase etc. for each transcript e.g., .Table 2ENote: The output file  of the extracted genomic material. Thus, harvesting SCN for downstream RNA extraction for a large number of biological replicates, covering distinct times of the day, may require few weeks.Bearing that in mind, this protocol describes the steps for transcriptome analysis at a resolution of 4 h during a day, and not for 2 days (48 h) or with higher resolution (2 h) to alleviate the risk of sample outliers and false-negatives in the rhythm detection. However, for significant rhythm detection from single circadian cycle, we chose a sampling interval of 4 h with increased number of biological replicates (n = 4) for each time-point, as per the sampling guidelines mentioned by Hughes et al. for genome-scale analysis of biological rhythms.Brain not properly frozen and foil wrapped (related to step 4).If the region to be dissected has come in direct contact with the foil, it will not be suitable for subsequent dissection. Discard the sample as per the institutional guidelines.The mouse brain sections are not even while vibratome sectioning (related to step 9).Make sure the 1% RNAse free PBS solution is chilled (0°C–4°C) before filling the metal tray. If the solution is not chilled, it will lead to thawing of the mouse brain which can result in uneven broken sections.SCN not visible under the dissection microscope (related to step 16).Adjust the reflective mirror at the base of the dissection microscope to light up the sample at different angles. Nuclei with denser cell bodies like the SCN will appear to be darker under direct reflected light, while its surrounding regions are lighter. Apart from the SCN it also works for other brain regions such as hippocampal formation, thalamic nuclei and zona inserta.RNA with poor integrity and quality (related to step 22).Inspect the integrity and purity of the extracted RNA. Proceed with the RNA extraction steps promptly, without any pause points, keeping the extracted RNA on ice to avoid RNA degradation.Unable to detect rhythmic gene expression using dryR .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Patrick M. Nolan (This study did not generate new unique reagents.


Antipsychotics are among the most widely prescribed molecules in psychiatry. Their sexual side effects are frequent, generally underestimated by clinicians and subjectively poorly tolerated by patients. They contribute to the significant non-compliance reported in treated patients. Most antipsychotics are non-selective and have actions on a multitude of receptors, both central and peripheral. Among these, the anti-dopaminergic action could have a deleterious effect on sexual function including spermatogenesis.The aim of our study is to report, according to the analysis of the collected data, the impact of the treatment with antipsychotic drugs on spermatogenesis and the counteraction of its consequences to a possible infertility in patients.The studies related to the treatment of DMDD were collected and analyzed. This study retrieved related articles from PubMed, SpringerLink, ScienceDirect, NCBI, CAIRN, and GOOGLE SCHOLAR. Use keywords “antipsychotics” AND “spermatogenesis” AND “infertility” AND “male” OR " hyperprolactinemia" AND “spermatogenesis” OR “risperidone” OR “olanzapine” OR " haloperidol" OR " fluphenazine" AND “spermatogenesis” AND “infertility” OR “psychotropics” AND “infertility” OR “spermatogenesis”.Antipsychotics are responsible, by blocking the secretion of dopamine in the central nervous system, for hyperprolactinemia, indirectly leading to hypogonadism. They are the pharmacological class most implicated in the occurrence of hyperprolactinemia. several studies have shown changes in the levels of sex hormones with alteration in the quality of the product of spermatogenesis, and others have demonstrated abnormalities in testicular architecture in rats after regular administration of doses of antipsychotics. thus, all antipsychotic molecules, whether classic or atopic, are likely to cause abnormalities in spermatogenesis.Antipsychotics are molecules with multiple indications in mental health, yet their adverse effects can become disabling or even irreversible. Sexual dysfunction and infertility are widespread among patients under antipsychotic treatment. The mechanisms of the effects of treatment with these molecules on spermatogenesis are poorly elucidated, since the vast majority of prospective studies are carried out on rats. However, this undesirable effect seems to be obvious and real.None Declared