5-Methylcytosine (m5C) is a type of RNA modification that exists in tRNAs and rRNAs and was recently found in mRNA. Although mRNA m5C modification has been reported to regulate diverse biological process, its function in adipogenesis remains unknown. Here, we demonstrated that knockdown of NOL1/NOP2/Sun domain family member 2 (NSUN2), a m5C methyltransferase, increased lipid accumulation of 3T3-L1 preadipocytes through accelerating cell cycle progression during mitotic clonal expansion (MCE) at the early stage of adipogenesis. Mechanistically, we proved that NSUN2 directly targeted cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA, a key inhibitory regulator of cell cycle progression, and upregulated its protein expression in an m5C-dependent manner. Further study identified that CDKN1A was the target of Aly/REF export factor (ALYREF), a reader of m5C modified mRNA. Upon NSUN2 deficiency, the recognition of CDKN1A mRNA by ALYREF was suppressed, resulting in the decrease of CDKN1A mRNA shuttling from nucleus to cytoplasm. Thereby, the translation of CDKN1A was reduced, leading to the acceleration of cell cycle and the promotion of adipogenesis. Together, these findings unveiled an important function and mechanism of the m5C modification on adipogenesis by controlling cell cycle progression, providing a potential therapeutic target to prevent obesity.

Alu RNA are implicated in the poor prognosis of several human disease states. These RNA are transcription products of primate specific transposable elements called Alu elements. These elements are extremely abundant, comprising over 10% of the human genome, and 100 to 1000 cytoplasmic copies of Alu RNA per cell. Alu RNA do not have a single universal functional role aside from selfish self-propagation. Despite this, Alu RNA have been found to operate in a diverse set of translational and transcriptional mechanisms. This review will focus on the current knowledge of Alu RNA involved in human disease states and known mechanisms of action. Examples of Alu RNA that are transcribed in a variety of contexts such as introns, mature mRNA, and non-coding transcripts will be discussed. Past and present challenges in studying Alu RNA, and the future directions of Alu RNA in basic and clinical research will also be examined.

Circular RNA is progressively reported to occur in various species including mammals where it is thought to be involved in the post-transcriptional regulation of gene expression, partly via interactions with microRNA. Here, we asked whether the circular topology causes functional differences to linear forms when interacting with short RNA strands in vitro and in human cells. Kinetic studies with human bladder cancer-derived synthetic circular RNA versus linear transcripts, respectively, with short oligoribonucleotides showed similar association rates for both topologies. Conversely, a substantial topology-related difference was measured for the activation entropy and the activation enthalpy of RNA-RNA annealing. This finding strongly indicates a significant difference of the mechanism of RNA-RNA interactions. To investigate whether these characteristics of circular RNA are biologically meaningful we performed transient transfection experiments with a microRNA-regulated expression system for luciferase in bladder cancer-derived cells. We co-transfected linear or circular RNA containing one microRNA binding site for the target-suppressing microRNA mlet7a. Here, the circular isoform showed a strongly increased competition with microRNA function versus linear versions. In summary, this study suggests novel topology-related characteristics of RNA-RNA interactions involving circRNA in vitro and in living cells.

The three-dimensional (3D) structure of RNA usually plays an important role in the recognition with RNA-binding protein. Along with the discovering of RNAs, several RNA databases are developed to study the functions of RNA based on sequence, secondary structure, local 3D structural motif and global structure. Based on RNA function and structure, different RNAs are classified and stored in SCOR and DARTS, respectively. The classification of RNA structures is useful in RNA structure prediction and function annotation. However, the SCOR and DARTS are not updated any more. In this study, we present an RNA classification database RR3DD based on RNA fold with the global 3D structural similarity. The RR3DD includes 13,601 RNA chains from PDB and mmCIF format structures which are classified into 780 RNA folds. The RNA chains from PDB and mmCIF format structures are aligned and clustered into 675 and 220 RNA folds, respectively. By analysing the RNA structure in RR3DD, we find that there are 11 clusters with more than 50 members. These clusters include rRNAs, riboswitches, tRNAs and so on. By mapping RR3DD into Rfam, we found that some RNAs without annotation by Rfam can be annotated through structural alignment. For example, we analysed tRNAs and found that tRNA were successfully grouped in RR3DD for which Rfam did not classify them into one family. Finally, we provide a web interface of RR3DD offering functions of browsing RR3DD, annotating RNA 3D structure and finding templates for RNA homology modelling.

As one of the most common forms of RNA modification, N6-methyladenosine (m6A) RNA modification has attracted increasing research interest in recent years. This reversible RNA modification added a new dimension to the post-transcriptional regulation of gene expression. In colorectal cancer (CRC), the role of m6A modification has been extensively studied, not only on mRNAs but also on non-coding RNAs (ncRNAs). In the present review, we depicted the role of m6A modification in CRC, systematically elaborate the interaction between m6A modification and regulatory ncRNAs in function and mechanism. Moreover, we discussed the potential applications in clinical.

Versatile RNA modifications play important roles in post-transcriptional regulations of gene expression, among which glycosylation modifications on small RNAs emerge as a novel clade whose characteristics need further interrogations. Here, we demonstrated that the sequence pattern around RNA glycosylation sites was not random and could be exploited for glycosylation site prediction. A machine learning predictor, GlyinsRNA, which integrated multiple RNA sequence representation encodings, was established. GlyinsRNA achieved AUROC (area under the receiver operating characteristic curve) of 0.7933 and 0.7979 in five-fold cross-validation and independent tests, respectively. GlyinsRNA was implemented as an online webserver, where both the predicted glycosylation sites and the overrepresented RNA-binding protein (RBP)-related motifs were annotated to facilitate the users. GlyinsRNA webserver is freely available at http://www.rnanut.net/glyinsrna.

Phage infection is one of the major threats to prokaryotic survival, and prokaryotes in turn have evolved multiple protection approaches to fight against this challenge. Various delicate mechanisms have been discovered from this eternal arms race, among which the CRISPR-Cas systems are the prokaryotic adaptive immune systems and phages evolve diverse anti-CRISPR (Acr) proteins to evade this immunity. Until now, about 90 families of Acr proteins have been identified, out of which 24 families were verified to fight against subtype I-F CRISPR-Cas systems. Here, we review the structural and biochemical mechanisms of the characterized type I-F Acr proteins, classify their inhibition mechanisms into two major groups and provide insights for future studies of other Acr proteins. Understanding Acr proteins in this context will lead to a variety of practical applications in genome editing and also provide exciting insights into the molecular arms race between prokaryotes and phages.

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5'-32P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.

TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein. We show that TUT4(7) ZnF2 contains two distinct RNA binding surfaces that are used in the interaction with different RNA nucleobases in different targets, i.e that this small domain encodes diversity in TUT4(7) selectivity and molecular function. Interestingly and unlike other well-characterized CCHC ZnFs, ZnF2 is not physically coupled to the flanking ZnF3 and acts independently in miRNA recognition, while the remaining CCHC ZnF of TUT4(7), ZnF1, has lost its intrinsic RNA binding capability. Together, our data suggest that the ZnFs of TUT4(7) are independent units for RNA and, possibly, protein-protein interactions that underlay the protein's functional flexibility and are likely to play an important role in building its interaction network.

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

By analysis of lncRNA expression profiles of macrophages in response to Mycobacterium tuberculosis (Mtb) infection, we identified novel highly expressed transcripts, unique in encompassing a protein coding transcript- Cytidine Monophosphate Kinase 2 (CMPK2) and a previously identified lncRNA- Negative Regulator of Interferon Response (NRIR). While these transcripts (TILT1, 2,3 - TLR4 and Infection induced Long Transcript) are induced by virulent Mtb as well as lipopolysaccharide (LPS) early, lack of/delayed expression in non-viable Mtb/BCG infected cells, respectively, suggest an important role in macrophage responses. The elevated expression by 3 hr in response to fast growing bacteria further emphasizes the importance of these RNAs in the macrophage infection response. Overall, we provide evidence for the presence of multiple transcripts that form a part of the early infection response programme of macrophages.Abbreviations: IFN: Interferon; NRIR: negative regulator of interferon response; CMPK2: cytidine/ uridine monophosphate kinase; LPS: lipopolysaccharide; LAM: Lipoarabinomannan; PIMs: Phosphatidylinositol Mannosides; TILT1, 2,3: TLR4 and Infection induced Long Transcript; TLR4: Toll-like receptor 4; Mtb: Mycobacterium tuberculosis; BCG: Mycobacterium bovis BCG; MDMs: human monocyte derived macrophages.

The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.

Non-membrane-bound compartments such as P-bodies (PBs) and stress granules (SGs) play important roles in the regulation of gene expression following environmental stresses. We have systematically and quantitatively determined the protein and mRNA composition of PBs and SGs formed before and after nutrient stress. We find that high molecular weight (HMW) complexes exist prior to glucose depletion that we propose may act as seeds for further condensation of proteins forming mature PBs and SGs. We identify an enrichment of proteins with low complexity and RNA binding domains, as well as long, structured mRNAs that are poorly translated following nutrient stress. Many proteins and mRNAs are shared between PBs and SGs including several multivalent RNA binding proteins that promote condensate interactions during liquid-liquid phase separation. We uncover numerous common protein and RNA components across PBs and SGs that support a complex interaction profile during the maturation of these biological condensates. These interaction networks represent a tuneable response to stress, highlighting previously unrecognized condensate heterogeneity. These studies therefore provide an integrated and quantitative understanding of the dynamic nature of key biological condensates.

In most bacteria, the three ribosomal RNAs (rRNAs) are encoded together in each of several near-identical operons. As soon as the nascent precursor rRNA emerges from RNA polymerase, ribosome assembly begins. This process entails ribosomal protein binding, rRNA folding, rRNA modification, and rRNA processing. In the model organisms Escherichia coli and Bacillus subtilis, rRNA processing results in similar mature rRNAs, despite substantial differences in the cohort of RNAses involved. A recent study of Flavobacterium johnsoniae, a member of the phylum Bacteroidota (formerly Bacteroidetes), revealed that helix H1 of 23S rRNA is absent from ribosomes, apparently a consequence of rRNA maturation. In this work, we mined RNA-seq data from 19 individual organisms and ocean metatranscriptomic samples to compare rRNA processing across diverse bacterial lineages. We found that mature ribosomes from multiple clades lack H1, and typically these ribosomes also lack an encoded H98. For all groups analysed, H1 is predicted to form in precursor rRNA as part of a longer leader-trailer helix. Hence, we infer that evolutionary loss of H98 sets the stage for H1 removal during 50S subunit maturation.

Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.

Upregulation of utrophin, the autosomal homologue of dystrophin, can compensate dystrophin deficiency in Duchenne Muscular Dystrophy (DMD) although the therapeutic success is yet to be achieved. The present study has identified Poly (C) binding protein 2 (PCBP2) as a post-transcriptional suppresser for the expression of utrophin-A, the muscle-specific utrophin isoform. This study confirms nuclear retention of utrophin-A mRNA in C2C12 cells, which is mediated by PCBP2. Further investigation demonstrates PCBP2-dependent nuclear retention of follistatin mRNA as well. Its involvement in nuclear retention of mRNA sheds light on a novel function of PCBP2 that makes utrophin-A mRNA less available in cytosol. PCBP2, therefore, may be a target to de-repress utrophin-A expression in DMD.

The well-established Shine-Dalgarno model suggests that translation initiation in bacteria is regulated via base-pairing between ribosomal RNA (rRNA) and mRNA. We used novel computational analyses and modelling of 823 bacterial genomes coupled with experiments to demonstrate that rRNA-mRNA interactions are diverse and regulate all translation steps from pre-initiation to termination. Previous research has reported the significant influence of rRNA-mRNA interactions, mainly in the initiation phase of translation. The results reported in this paper suggest that, in addition to the rRNA-mRNA interactions near the start codon that trigger initiation in bacteria, rRNA-mRNA interactions affect all sub-stages of the translation process (pre-initiation, initiation, elongation, termination). As these interactions dictate translation efficiency, they serve as an evolutionary driving force for shaping transcripts in bacteria while considering trade-offs between the effects of different interactions across different transcript regions on translation efficacy and efficiency. We observed selection for strong interactions in regions where such interactions are likely to enhance initiation, regulate early elongation, and ensure translation termination fidelity. We discovered selection against strong interactions and for intermediate interactions in coding regions and presented evidence that these patterns maximize elongation efficiency while also enhancing initiation. These finding are relevant to all biomedical disciplines due to the centrality of the translation process and the effect of rRNA-mRNA interactions on transcript evolution.

The field of epitranscriptomics is rapidly developing. Several modifications (e.g. methylations) have been identified for different RNA types. Current evidence shows that chemical RNA modifications can influence the whole molecule's secondary structure, translatability, functionality, stability, and degradation, and some are dynamically and reversibly modulated. miRNAs, in particular, are not only post-transcriptional modulators of gene expression but are themselves submitted to regulatory mechanisms. Understanding how these modifications are regulated and the resulting pathological consequences when dysregulation occurs is essential for the development of new therapeutic targets. In humans and other mammals, dietary components have been shown to affect miRNA expression and may also induce chemical modifications in miRNAs. The identification of chemical modifications in miRNAs (endogenous and exogenous) that can impact host gene expression opens up an alternative way to select new specific therapeutic targets.Hence, the aim of this review is to briefly address how RNA epitranscriptomic modifications can affect miRNA biogenesis and to summarize the existing evidence showing the connection between the (de)regulation of these processes and disease settings. In addition, we hypothesize on the potential effect certain chemical modifications could have on the potential cross-kingdom journey of dietary plant miRNAs.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.

From yeast to humans, pre-mRNA splicing occurs mainly co-transcriptionally, with splicing and transcription functionally coupled such that they influence one another. The recruitment model of co-transcriptional splicing proposes that core members of the transcription elongation machinery have the potential to influence co-transcriptional spliceosome assembly and pre-mRNA splicing. Here, we tested whether the transcription elongation kinases Bur1 and Ctk1 affect co-transcriptional spliceosome assembly and pre-mRNA splicing in the budding yeast Saccharomyces cerevisiae. In S. cerevisiae, Ctk1 is the major kinase that phosphorylates serine 2 of the carboxy-terminal domain of the largest subunit of RNA polymerase II, whilst Bur1 augments the kinase activity of Ctk1 and is the major kinase for elongation factor Spt5. We used the auxin-inducible degron system to conditionally deplete Bur1 and Ctk1 kinases, and investigated the effects on co-transcriptional spliceosome assembly and pre-mRNA splicing. Depletion of Ctk1 effectively reduced phosphorylation of serine 2 of the carboxy-terminal domain but did not impact co-transcriptional spliceosome assembly or pre-mRNA splicing. In striking contrast, depletion of Bur1 did not reduce phosphorylation of serine 2 of the carboxy-terminal domain, but reduced Spt5 phosphorylation and enhanced co-transcriptional spliceosome assembly and pre-mRNA splicing, suggesting a role for this kinase in modulating co-transcriptional splicing.

In plants, recent studies have revealed that N6-methyladenosine (m6A) methylation of mRNA has potential regulatory functions of this mRNA modification in many biological processes. m6A methyltransferase, m6A demethylase and m6A-binding proteins can cause differential phenotypes, indicating that m6A may have critical roles in the plant. In this study, we depicted the m6A map of sea buckthorn (Hippophae rhamnoides Linn.) transcriptome. Similar to A. thaliana, m6A sites of sea buckthorn transcriptome is significantly enriched around the stop codon and within 3'-untranslated regions (3'UTR). Gene ontology analysis shows that the m6A modification genes are associated with metabolic biosynthesis. In addition, we identified 13,287 different m6A peaks (DMPs) between leaf under drought (TR) and control (CK) treatment. It reveals that m6A has a high level of conservation and has a positive correlation with mRNA abundance in plants. GO and KEGG enrichment results showed that DMP modification DEGs in TR were particularly associated with ABA biosynthesis. Interestingly, our results showed three m6A demethylase (HrALKBH10B, HrALKBH10C and HrALKBH10D) genes were significantly increased following drought stress, which indicated that it may contributed the decreased m6A levels. This exhaustive m6A map provides a basis and resource for the further functional study of mRNA m6A modification in abiotic stress.

Breast cancer (BC) as a leading cause of cancer death among women, exhibits a wide range of genetic heterogeneity in affected individuals. Satisfactory management of BC depends on early diagnosis and proper monitoring of patients' response to therapy. In this study, we aimed to assess the relation between the expression patterns of blood-based microRNAs (miRNAs) with demographic characteristics of the patients with BC in an attempt to find novel diagnostic markers for BC with acceptable precision in clinical applications. To this end, we performed comprehensive statistical analysis of the data of the Cancer Genome Atlas (TCGA) database and the blood miRNome dataset (GSE31309). As a result, 21 miRNAs were selected for experimental verification by quantitative RT-PCR on blood samples of 70 BC patients and 60 normal individuals (without any lesions or benign breast diseases). Statistical one-way ANOVA revealed no significant difference in the blood levels of the selected miRNAs in BC patients compared to any lesions or benign breast diseases. However, the multi-marker panel consisting of hsa-miR-106b-5p, -126-3p, -140-3p, -193a-5p, and -10b-5p could detect early-stages of BC with 0.79 sensitivity, 0.86 specificity and 0.82 accuracy. Furthermore, this multi-marker panel showed the potential of detecting benign breast diseases from BC patients with 0.67 sensitivity, 0.80 specificity, and 0.74 accuracy. In conclusion, these data indicate that the present panel might be considered an asset in detecting benign breast disease and BC.

Exportin 5 (Exp5, XPO5) is a nuclear export factor that functions in the microRNA (miRNA) biogenesis pathway to transport precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm. Most of our current understanding of the Exp5 and pre-miRNA interaction is based on the investigation of how Exp5 binds to human pre-miR-30a (pre-miR-30 for short). As there are hundreds of human miRNA genes, how representative pre-miR-30 is, whether or how Exp5 interacts with distinct cargoes differentially, or whether Exp5 regulates miRNA expression, is unknown. Here we examined and compared the interactions between Exp5 and 157 human pre-miRNAs. We found that Exp5 binds distinct pre-miRNAs with modest variations in efficiencies, with the 3' overhang and the apical loop in pre-miRNAs being the two major discriminating factors. Exp5 binding efficiencies do not significantly correlate with endogenous miRNA expression, suggesting that Exp5 activity does not contribute to differential miRNA expression in vivo. Nonetheless, in human cells with reduced Exp5 levels, preferential Exp5 binding correlated with miRNA expression changes. Thus, our study provides a global picture of how Exp5 interacts with human pre-miRNAs and its role in regulating miRNA expression.Abbreviations: Exp5: Exportin 5; miRNA: microRNA; pri-miRNA: primary microRNA; pre-miRNA: precursor microRNA; nt: nucleotide.