The development of new sequencing technologies in the post-genomic era has accelerated the identification of causative mutations of several single gene disorders. Advances in cell and animal models provide insights into the underlining pathogenesis, which facilitates the development and maturation of new treatment strategies. The progress in biochemistry and molecular biology has established a new class of therapeutics—the short RNAs and expressible long RNAs. The sequences of therapeutic RNAs can be optimized to enhance their stability and translatability with reduced immunogenicity. The chemically-modified RNAs can also increase their stability during intracellular trafficking. In addition, the development of safe and high efficiency carriers that preserves the integrity of therapeutic RNA molecules also accelerates the transition of RNA therapeutics into the clinic. For example, for diseases that are caused by genetic defects in a specific protein, an effective approach termed “protein replacement therapy” can provide treatment through the delivery of modified translatable mRNAs. Short interference RNAs can also be used to treat diseases caused by gain of function mutations or restore the splicing aberration defects. Here we review the applications of newly developed RNA-based therapeutics and its delivery and discuss the clinical evidence supporting the potential of RNA-based therapy in single-gene neurological disorders. Over the past 30 years, knowledge of neurogenetics has evolved considerably. Advances in sequencing technology and molecular medicine have led to the identification of many genes that are involved in neurological diseases. Functional studies have revealed new insights in biochemistry and biological processes that are associated with disease pathophysiology. Based on these molecular insights and the technology breakthrough in therapeutic developments, the treatments for previously incurable diseases were proven to be feasible and will accelerate in the coming decade. Given the progress in genetic diagnosis and understanding of the pathophysiology of neurological disorders, especially single gene disorders, we aim to summarize the recent development of targeted ribonucleic acid (RNA)-based therapy and its deliveries in this review.The human genome contains three billion deoxyribonucleotides and is organized into 23 pairs of chromosomes ,2. GenesA single nucleotide change can result in a silent, missense, or nonsense mutation. A silent mutation does not change the amino acid residue, but a missense mutation does. Depending on the impact of mutated amino acid residue, a missense mutation can cause haploinsufficiency, a gain of function, or a dominant negative effect. A nonsense mutation can lead to a stop codon which results in premature termination of protein translation. A frameshift mutation that is caused by insertion, deletion, or rearrangement of the DNA sequences can also disrupt the entire reading frame of a gene as well as introduce a premature stop codon, which results in a severe dysfunctional protein. Haploinsufficiency or loss of function that is caused by the truncated protein is frequently encountered in tumor suppressor proteins e.g., p53 and PTEN)  and PTEN or enzymThe molecular mechanisms for the diseases include loss of function, haploinsufficiency, and toxic gain of function. Understanding the molecular pathology would help in designing treatment strategies. Recently, the advances in precision medicine and targeted therapy have been nourished by the progress in sequencing technologies and biostatistical analysis. The alteration of a disease-associated gene in either the concentration or its sequence attracted attention in recent years. The manipulation at the transcription level becomes feasible with the introduction of the eukaryotic expression system and RNA interference technology. In addition to the direct manipulation on the level of a specific transcript, the idea to modify the splicing process to preserve the partial function of the very protein which is pivotal to physiological needs has been introduced. Furthermore, the newly designed nanocarriers to facilitate the delivery and release of the nucleic acids without causing immune responses has also made progress in recent years. Thus, RNA therapy to single-gene neurological disorders in routine clinical practice is on the horizon.The use of nucleic acid therapy to treat monogenetic diseases is on the horizon of genomic medicine. Based on the preclinical studies in cellular and animal models, the emergence of therapeutic messenger RNAs (mRNAs) and short RNAs was proven to be a new class of medicine in a spectrum of diseases , such asFor diseases that are caused by haploinsufficiency or loss of function, replacing the defective protein is a straightforward treatment strategy. Proteins that are synthesized by bacteria or cultured cells, or synthetic peptides are commonly used to supply or replace the defective proteins. Even though the use of recombinant protein extended the arsenal of treating some single gene neurological diseases, there are many drawbacks including the high cost, potential immunogenicity, and only suitable for some disease entities . RNA-basAn alternative to use a viral vector for defective gene replacement is to introduce chemically-modified mRNA directly into the cells. This approach has been adapted in oncology as well as cardiovascular and infectious diseases ,14. The 1-methylpseudouridine or 5-methoxyuridine can also be used to increase the protein yield. Moreover, the modified mRNA can reduce the activation of immune RNA sensors such as the retinoic acid-inducible gene I and the Toll-like receptors [Structurally, the mRNA is composed of a 5′ cap followed by a 5′ untranslated region (UTR), a protein coding region, a 3′ UTR, and a poly (A) tail. The 5′ cap, UTR regions, as well as the poly (A) tail are important for stabilizing the mRNA ,18. Modieceptors . The inaIn addition to full-length mRNA for protein replacement therapy, short single stranded oligonucleotides or chimeric RNA-DNA have been explored as a therapeutic modality . Single-Based on the mode of action, there are three major types, i.e., gapmers, steric blocking, and CpGs. Gapmers are 16–20 nucleotides (nts) oligomers with a central stretch of 8–10 DNA nts flanked by a stretch of 4–5 nts on each side with modifications on the sugar rings. The sugar modifications can increase binding affinity, avoid or decrease immune activation, and increase resistance to nuclease. The PS bonds in the sugar ring of gapmers can enhance the stability and increase binding to plasma proteins . GapmersSteric-blocking ASOs are single-stranded oligonucleotides which are 15~30 nts in length. Both the sugar ring and the backbone of the nucleotides can be chemically modified. Similar to gapmer ASOs, the chemical modifications are intended to increase the nuclease resistance and binding affinity, as well as to reduce immune responses. Steric-blocking ASOs bind to the target RNA via sequence-specific base-pairing. The complementary pairing hinders the binding of trans-acting factors such as small nuclear RNAs, microRNAs, long non-coding RNAs, or RNA binding proteins, to their cognate sequences. The pairing between the steric-blocking ASOs and the target RNA also prevents the formation of proper RNA secondary structure ,29. The USH2A gene encoding usherin protein contains multiple repetitive domains. A deletion mutation in exon 13 of the USH2A gene occurs in 30% of USH2 patients. Pendse et al. hypothesized that the removal of one or more of the repetitive domains that is encoded by mutant exon(s) may restore the normal function of the USH2A protein [A different application for steric-blocking ASO is exon-skipping to eliminate the exons with a missense mutation in the final mature mRNA. This strategy was first proposed for Emery-Dreifuss muscular dystrophy, a disease that is caused by mutations in the lamin A/C gene (LMNA) . There i protein . An antiRecently, a novel approach named targeted augmentation of nuclear gene output (TANGO) was introduced to use antisense oligonucleotides to modulate non-productive splicing events and enhance the level of functional transcript/protein . Lim et In summary, therapies utilizing single-stranded oligonucleotides represent a platform of precision medicine to treat genetic diseases that were once deemed untreatable. The innovation on the modification chemistries in the ASO drugs makes them stable, safe, and effective to be used in patients. Together with targeted delivery, ASO-based therapeutics are expanding the breadth in precision medicine and paving the way for clinical applications in previously untreatable genetic diseases. Effectively delivering the therapeutic agents to the target site is another important issue. For example, there are blood-brain and blood-nerve barriers that separate the nervous tissues from the systemic circulation. The next section will discuss the newly developed drug carriers which overcome these obstacles and can release the drug at or nearby the disease site.Anatomical and metabolic barriers between the brain parenchyma and the peripheral blood circulation maintain the proper activities of the central nerve system (CNS). These barriers include cerebrospinal fluid (CSF), choroid plexus, and the blood brain barrier (BBB). The BBB is a protective anatomic structure that prevents the brain from direct contact with the blood , an impoTo move molecules across BBB is usually through either a carrier-mediated or a receptor-mediated transport (RMT) system. An array of RMT transporters, receptors, and channels on endothelial cells and pericytes are involved in the process . Thus, tTo adopt a new therapeutic entity into the clinic, several key data including toxicity profile, ADME  characteristics, and dosage need to be generated. For CNS drugs, the BBB is an additional factor that needs to be addressed; therefore, the delivery system is especially important for therapeutics that are used in neurological diseases. Though drugs can be delivered to the brain by local injection, via a catheter, or direct administration following an invasive surgery ,53, thesThe growing field of nanoscale delivery systems for therapeutics is the main focus of basic research . NanocarThe lipid nanoparticles  are the pioneer of this rapidly evolving field. There is a long list of attributes and advantages of lipid-based drug delivery systems such as controlled and targeted drug release, pharmaceutical stability, capability for incorporation of both lipophilic and hydrophilic drugs, biodegradability and biocompatibility, low-risk profile, better drug absorption, and lowering the therapeutic dose, etc. The structural modifications of lipid-based nanoparticles enable the delivery carrier to safely and effectively pass through the BBB . VehicleOne of the key elements for RNA-based therapy is a carrier that can protect the RNA from nuclease degradation and penetrate the BBB with adequate bioavailability. Lipid nanoparticles are usually composed of phospholipids, sterols, and ionizable lipids. Adding polyethylene glycol (PEG)—conjugated lipids can reduce the capture by phagocytes after systemic administration. In clinical trials, the liver is the main organ for the distribution of lipid nanoparticles. By interacting with the low-density lipoprotein (LDL) receptor, apolipoprotein E, and other opsonins, the nanoparticles are internalized into the hepatocytes . With chSolid lipid nanoparticles (SLN) have recently gained more attention as a drug delivery system . SLNs arExtracellular vesicles (EV) were discovered 30 years ago . RecentlThe cargos that are carried by EVs include metabolites, DNA, proteins, and RNA ,91,92,93The viral genome can be modified as a carrier to insert genetic materials into cells. As an example, using the gamma-retroviral vector to transduce hematopoietic stem cells to treat adenosine deaminase deficiency-induced severe combined immunodeficiency was approved by the EMA in 2016 . The FDAIn this section, a few RNA therapeutic agents targeting single-gene neurological disorders will be introduced. These neurological disorders involve the CNS and PNS as well as multi-systems, although they are caused by mutations from single genes. Given the progress in RNA therapy, a few RNA drugs have been approved by FDA in the US . DespiteHereditary amyloidosis is a rare, life-threatening, autosomal dominant, multi-organ disease that is characterized by amyloid deposits in multiple organs. Mutations in several genes are involved in hereditary amyloidosis and the most common one is the mutations in the transthyretin (TTR) gene . The priIn 2012, Su et al. described that mice without TTR expression are viable . Thus, sAcute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder that is caused by the deficiency of porphobilinogen deaminase (PBGD), an enzyme that is involved in the synthesis of heme . The δ-aThe infusion of hemin and loading of carbohydrates are the frequently used treatments for AIP. The supply of hemin can restore the heme pool in the liver and also suppress the ALAS1 expression . About 5ALAS1 transcript [An alternative treatment strategy utilizes a short interfering RNA to reduce the level of anscript . The appanscript ,136,137.® (2′-O-methoxyethylphosphorothioate)  and Eteplirsen®  [As mentioned earlier, utilizing ASO can specifically decrease the level of the targeted protein. Another class of oligonucleotide therapeutics are single-stranded splice-switching oligonucleotides (SSOs) which target the pre-mRNA of interest in the nucleus and alter its splicing process to produce a functional protein . In 2016de, PMO)  for the de, PMO) .®) in 2016 for the treatment of all types of SMA [Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is also the leading inherited monogenetic cause of death in infants. The motor neuron degeneration in SMA causes marked weakness and atrophy in the limbs and bulbar muscles . A delets of SMA .®) was approved by the FDA in 2019 [In addition to the SSO to block the aberrant splicing, gene-replacement therapy (Onasemnogene abeparvovec) that is based on adeno-associated virus serotype 9 (AAV9) to provide the full-length SMN1 protein has been designed. Increased SMN protein expression following the treatment has successfully prevented the death of motor neurons with the improvement of neuromuscular functions in the affected children. The phase 3 trial provides promising outcomes and Onasemnogene abeparvovec  is one of the most common inherited muscular disorders. DMD is caused by the mutations in a large dystrophin coding gene (DMD), 3685 amino acids long from 79 exons spanning more than 2Mb in the genome . Most ofAs aforementioned, microsatellite repeat expansion (MRE) in both coding and non-coding regions of the genome has been associated with many neurological disorders, such as Huntington’s disease (HD), spinocerebellar ataxia, Friedreich ataxia, myotonic dystrophy, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) . The halhttps://www.ninds.nih.gov/Disorders/All-Disorders/Huntingtons-Disease-Information-Page, accessed on 18 November 2019) have been used to relieve the symptoms. The expansion of the CAG trinucleotide repeats in the HTT gene leads to an extended polyglutamine in HTT which promotes protein aggregation—the pathogenic culprit of HD.HD is an autosomal dominant neurodegenerative disorder resulting from a mutation in the human huntingtin (HTT) gene. The clinical features include dementia, depression, schizophrenia, hyperkinetic movements such as chorea and athetosis, oculomotor apraxia, bipolar disorders, and sometimes suicidal events . There ihttps://www.globenewswire.com/news-release/2021/03/29/2201081/0/en/Wave-Life-Sciences-Provides-Update-on-Phase-1b-2a-PRECISION-HD-Trials.html, accessed on 10 November 2021). In addition to ASO, another strategy is using microRNA to recruit HTT mRNA to the RNA-induced silencing complex (RISC) which leads to the degradation of the transcripts. In animal studies, AAV9 viral particles carrying miRNA have been designed and delivered via direct intrastriatal administration resulting in a therapeutic benefit [Recently, an RNA-based target therapy has been developed utilizing ASOs which binds to the HTT transcript followed by the activation of an RNase H-mediated HTT mRNA degradation . The cop benefit ,170,171 Lysosomal storage disorders (LSD) are a group of rare diseases and more than two thirds of LSD patients present CNS involvement. The defect of lysosomal proteins in LSD results in an accumulation of nonmetabolized pathogenic products within neurons or other cells. For example, Fabry disease is caused by the accumulation of globotriaosylceramide in cells due to alpha-galactosidase A (GLA) deficiency. The accumulation of glucosylceramide due to the lack of beta glucocerebrosidase (GBA) is caused the Gaucher disease. The LSD-induced neurodegeneration may start as early as the first decade of life, and, once triggered, the progression is unrelenting . A few cOne of the approved therapies for LSD is an enzyme replacement therapy (ERT), by providing a recombinant protein or an enzyme to replace the defective one. Some positive results for ERT in Gaucher, Fabry, and Pompe diseases have been reported ,176,177.Different synthetic nanoparticles, from electrolytic complexes to liposomes and aggresomes have been tested to deliver small RNAs and mRNAs within the last decade . ExtraceTo generate high-purity therapeutic grade RNA molecules with proper chemical modifications is much faster and less expensive than the production of either traditional small molecule drugs or recombinant proteins. In addition, the manufacturing process is independent of the RNA sequence. Compared to DNA-based gene therapy, RNA has a superior safety profile and the regulatory requirements are easy to follow. Moreover, RNA doesn’t integrate into the host genome . There aRecently, hospital-based RNA therapeutic development has successfully demonstrated the feasibility of generating novel nucleic acid drugs for patients. One of the examples is the investment of National Children’s hospital into cGMP facilities to introduce gene therapies for SMA (onasemnogene abeparvovec) and DMD (golodirsen). Thus, hospital-based programs will promote the development and implementation of personalized medicines. It is reasonable that the hospital generates the cGMP grade RNA therapy and quickly delivers it to the patient, since manufacturing of the drug at such a small scale is rarely justified financially by the big pharma companies. Damase et al. described their effort to quickly develop new constructs in manufactures research and clinical grade RNA therapeutics . Their tAdvances in molecular diagnosis and the development of RNA-based medications reveal a great potential in precision therapy for neurological disorders. One of the major advantages of RNA-based therapy is its flexibility as it can target both coding and non-coding RNAs. It can also work on previously undruggable targets, for example, the knockdown of TTR expression by ASOs. RNA therapeutics can be designed rapidly. As soon as the sequence is established, the next step is to modify the sequence to minimize the degradation and enhance its binding stability.For some RNA-based therapies, for example, enzyme replacement therapy, it can have long-lasting effects compared to the traditional small molecule-based drugs where the therapeutic effects are completely governed by its ADME profile . To develop long-lasting RNA-based medications to treat or cure patients with a monogenic disease since birth remains attractive to researchers. Apart from the RNA drugs, the delivery system plays a critical role in the success of RNA-based therapies, especially for treatments that are targeting the brain. The recent development of lipid-based nanocarriers and extracellular vesicles provides a possibility to effectively deliver therapeutics passing through the blood brain barrier. In summary, we are witnessing the development of RNA therapies to treat monogenic diseases. Further refinement to optimize the design of therapeutic RNA molecules and the delivery system may shed light on how to treat previously untreatable hereditary neurological diseases.


The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm. RNA silencing, a small RNA (sRNA)-mediated sequence-specific mechanism, is a primary antiviral response of host plants against viral infection. Argonaute 1 (AGO1), an effector protein of RNA silencing, associates with sRNAs to degrade viral RNAs or silence the genes involved in biotic stresses in cytoplasm; such functions are promoted by its translocation between the nucleus and cytoplasm. To survive host RNA silencing, cucumber mosaic virus (CMV) encodes an RNA silencing suppressor (RSS), 2b protein (2b). 2b plays multiple roles in suppressing RNA silencing by binding to either sRNAs or AGOs, in the nucleus and the cytoplasm. However, several previous reports suggest that 2b exerts its RSS activity mainly when it is in the cytoplasm. Here, we found that the serine residue at position 28 is phosphorylated, and its phosphorylation negatively regulated the RSS activity of 2b by regulating 2b’s nuclear/nucleolar localization. Furthermore, we also found that 2bs were dephosphorylated in the nucleus and exported to the cytoplasm in an exportin-dependent manner. Because 2b’s transport between the nucleus and cytoplasm is similar to that of AGOs, 2b seems to work in cells as if it is manipulating its nucleocytoplasmic trafficking to track down its targets in the RNA silencing pathway. The nuclear envelope is a physical barrier that regulates dynamic compartmentation of proteins between the nucleus and cytoplasm . BecauseIn contrast, the export of a protein from the nucleus is generally mediated by nuclear export signals (NESs). NESs are mostly leucine-rich motifs that provide an epitope for the interaction with a transporter of nuclear export, exportin ,12. MuchCucumber mosaic virus (CMV) has three positive-sense, single-stranded genomic RNAs (RNAs 1–3) ; it encoin vivo or in vitro, neutralizing the siRNA- or miRNA-mediated post-transcriptional gene silencing (PTGS) S28 was phosphorylated by PKC . Curiousin planta, we examined in vivo phosphorylation of these serine residues. For the phosphorylation assay, we used the CMV-H1 RNA 2 vector in which the entire 2b open reading frame (ORF) was deleted and a multiple cloning site (MCS) was inserted; a stop codon for 2a was added before MCS  are phosphorylated in planta.To confirm the phosphorylation of S28, S40 and S42 fore MCS  48]. Th. Thin plfore MCS . Our wesfore MCS . Using afore MCS . PhosphoN. benthamiana by agroinfiltration. The wild-type 2b-GFP was detected in the nucleus and nucleolus as previously reported ) using a Slide-A-Lyzer MINI Dialysis Unit . Purified proteins were incubated in the kinase reaction buffer  containing 37 nM ATP, 0.25% Complete Mini Protease Inhibitor Cocktail  and 0.5 μL of protein kinase C . The in vitro phosphorylated proteins were then separated by SDS-PAGE and detected using the ProQ-Diamond Phosphoprotein Gel Stain kit .Seven putative phosphorylation sites including one threonine and six serines were predicted by the NetPhos v2.0 program (escribed . The extN. tabacum plants at 6 days post inoculation. Phosphorylated proteins were concentrated using the TALON PMAC phosphoprotein enrichment kit (Clontech) according to the manufacturer’s instructions  were homogenized in 1 mL of the lysis buffer  Triton-X 100, 1 mM PMSF, 10% (v/v) glycerol) containing 1X protease inhibitor cocktail (Sigma Aldrich) S28, [A]S40/S42 and [A]S28/S40/S42 were created using the primers in t-test . (E) Symptoms on upper leaves of the plants 21 days after inoculation (dpi) with either CMV:[A]S40/S42 or CMV:[A]S28/S40/S42. (F) Relative accumulation levels of CMV RNA 3 and 4 in the plants infected with CMV:[A]S40/S42 or CMV:[A]S28/S40/S42 at 21 dpi. Tissues were collected from the leaves in t-test (**P < 0.01).(A) Mutants with alanine substitutions at various putative phosphorylation sites were created, and CMV RNA 2 constructs with those 2b mutants were prepared by inserting the 2b fragments into the CMV H1 vector. (B) Symptom severity on (TIF)Click here for additional data file.S13 Fig(A) RSS activity of 2b, S77/85/87A and the C-terminal deletion mutants. A series of C-terminal deletion mutants were created using S77/85/87A as the background (B), and their RSS activity was examined as described in (TIF)Click here for additional data file.S14 Fig2, 2 mM 2-mercaptoethanol), and the cytoplasmic fraction was separated by a series of differential centrifugations. The supernatant after the final centrifugation was used for western blot analysis.Agroinfiltrated leaf tissues were homogenized in extraction buffer Click here for additional data file.S15 Fig(A) Based on the 2b-siRNA binding model in (TIF)Click here for additional data file.S16 FigTo confirm that the 2b’s mutations are maintained during viral infection, the entire 2b sequences were amplified by RT-PCR from the infected plants at 15 dpi. Their sequences were analyzed by the BigDye terminator method. The wave raw data of the sequencing analyses were shown. Each codon of the mutations is indicated in red bars.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


In order to study the influence of RNA editing sites on lactation and mammary gland development process in yaks, we comprehensively characterized the RNA editome of the yak mammary gland during the lactation period and dry period by using the transcriptome and genome sequencing data. The results revealed 82,872 nonredundant RNA editing sites, 14,159 of which were differentially edited between the lactation period and dry period. Enrichment analysis showed that the genes harboring differential editing sites were mainly associated with mammary gland development-related pathways, such as MAPK pathway, PI3K-Akt pathway, FoxO signaling pathway, GnRH signaling pathway, and focal adhesion pathway. Our findings offer some novel insights into the RNA editing function in the mammary gland of yaks.n = 2) and dry period . Totally, 82,872 unique RNA editing sites were identified, most of which were detected in the noncoding regions with a low editing degree. In the coding regions (CDS), we detected 5235 editing sites, among which 1884 caused nonsynonymous amino acid changes. Of these RNA editing sites, 486 were found to generate novel possible miRNA target sites or interfere with the initial miRNA binding sites, indicating that RNA editing was related to gene regulation mediated by miRNA. A total of 14,159 RNA editing sites (involving 3238 common genes) showed a significant differential editing level in the LP when compared with that in the DP through Tukey’s Honest Significant Difference method (p < 0.05). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, genes that showed different RNA editing levels mainly participated in pathways highly related to mammary gland development, including MAPK, PI3K-Akt, FoxO, and GnRH signaling pathways. Collectively, this work demonstrated for the first time the dynamic RNA editome profiles in the mammary gland of yaks and shed more light on the mechanism that regulates lactation together with mammary gland development.The mammary gland is a complicated organ comprising several types of cells, and it undergoes extensive morphogenetic and metabolic changes during the female reproductive cycle. RNA editing is a posttranscriptional modification event occurring at the RNA nucleotide level, and it drives transcriptomic and proteomic diversities, with potential functional consequences. RNA editing in the mammary gland of yaks, however, remains poorly understood. Here, we used REDItools to identify RNA editing sites in mammary gland tissues in yaks during the lactation period (LP,  RNA editing refers to a newly discovered mechanism that alters the RNA sequence itself without altering its genomic DNA (gDNA) through nucleotide deletion, insertion, and substitution . These tBos grunniens), a multipurpose livestock, is widely distributed in the Qinghai-Tibetan plateau and adjacent regions [Yak . The MGIEasy Universal DNA Library Prep Kit  was adopted for constructing DNA libraries in accordance with the BGI’s standard preparation protocol. We sequenced the library as 150 base-pair (bp) paired-end runs by using MGISEQ2000  at Frasergen Bioinformatics Co., Ltd. .https://broadinstitute.github.io/picard, (accessed on 30 October 2017). Additionally, we used Hisat2 (v2.1.0) [After removing ploy-N, low-quality bases, and contaminating adapter molecules from the raw data, we aligned DNA clean reads to the domestic yak genome sequence (LU_ Bosgru_v3.0) by BWA (v0.7.17) . Samtool(v2.1.0)  to map R(v2.1.0)  was usedThe REDItoolDnaRna.py script (REDItools)  was used−5 and identity more than 85% to be the conserved editing sites. This study obtained known human RNA editing sites based on REDIportal  databases [For conservation analysis, we aligned the 25-bp sequence in upstream and downstream of candidate RNA editing sites against 50-bp flanking regions of the reported human sites by employing the NCBI BLAST tools blastn. We deemed alignments with e-values < 1 × 10atabases . We usedRegarding the editing sites in mRNA 3′ untranslated regions (UTRs), we adopted RNAhybrid   and miRap < 0.05) between the LP and DP. In order to illustrate RNA editing sites related to lactation and mammary gland growth at the genome-wide scale, we used Tukey’s Honest Significant Difference method to identify RNA-edited sites as significantly different , RNA editing sites within miRNA binding sites may influence the recognition between miRNA and target mRNA. Around 1.63% of the RNA editing sites were located within 3′ UTR, which included 486 sites that were predicted to change miRNA binding capacity. Among these 486 RNA editing sites, 294 produced 449 novel possible miRNA binding sites, and 237 destroyed 363 miRNA target sites . This st−5), 74 conserved RNA editing sites were identified in 36 genes (SON), forkhead box O3 (FOXO3), ArfGAP With RhoGAP domain, ankyrin repeat and PH domain 1 (ARAP1), Nei-like DNA glycosylase 1 (NEIL1), THO complex 1 (THOC1), H2A clustered histone 20 (H2AC20), chromatin target Of PRMT1 (CHTOP), cyclin-dependent kinase 13 (CDK13), and insulin-like growth factor binding protein 7 (IGFBP7). Of these, editing sites in SON, NEIL1, CDK13, and IGFBP7 genes may lead to missense mutations. To examine the conservation of RNA editing sites in human and yak, this study performed cross-species comparison by conducting BLAST analysis. When strict thresholds were applied to the results   a. On the < 0.05) . The GO  < 0.05) . In the  < 0.05) a. As sug < 0.05) .RNA editing is an important source of molecular diversity that increases transcript flexibility and diversity by altering specific nucleotides within RNA posttranscriptionally. With the rapid development and application of NGS technologies, numerous RNA editing sites have been characterized in the genome of diverse animal species. To date, the role of RNA editing in the development of the mammary gland is ambiguous. In the present study, we comprehensively identified the genome-wide RNA editing events in the yak mammary gland.In this study, we identified 82,872 editing sites in yak mammary tissues at the LP and DP. Notably, the number of editing sites was significantly different between the LP and DP. Compared to the LP, a higher number of editing sites was identified in the DP, which indicated higher RNA editing activity. Twelve types of RNA editing were detected, among which the proportion of A-to-G was the highest. Among all the RNA editing types, A-to-G and C-to-T were identified to be the canonical RNA editing events, whereas the remaining RNA editing types were the non-canonical ones . The A-tConsistent with previous studies on cattle and pig ,10, our BRCA1), a breast cancer susceptibility gene, is involved in lobular-alveolar development in the mammary gland [LTF) encodes a major iron-binding protein in milk, which has both bacteriostatic and bactericidal activity [NRIP1), also known as RIP140, is a co-regulator for transcription factors that regulate mouse mammary gland development [cis-regulatory impact on gene expression by influencing mRNA degradation and stabilization.Annotation of the edited genes with missense mutations in the mammary gland revealed that some of the genes were associated with mammary gland development and milk traits. For instance, BRCA1 DNA repair associated  is an amplification factor of the PI3K/AKT pathway, which is considered the regulatory factor for mammary gland growth. IGF1 overexpression has been previously shown in pregnant or lactating mice, which leads to delayed mammary epithelial cell apoptosis in the declining phase of lactation [IGF1 gene is associated with milk fat and protein yields [INSIG1), an endoplasmic reticulum membrane protein, has an important function in regulating lipid synthesis [INSIG1 [STAT3) belongs to the STAT protein family and participate in the mouse mammary epithelial cell apoptosis [FGF10) regulates mammary gland development and homeostasis by acting through the fibroblast growth factor receptor 2B (FGFR2B) [Our genome-wide differential RNA editing analyses yielded a set of 14,159 sites that were significantly differentially edited between the LP and DP. The KEGG analysis showed that the genes with differential editing level were enriched in some critical pathways related to lactation and the mammary gland, which included focal adhesion pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, FoxO signaling pathway, GnRH signaling pathway, and ECM-receptor interaction pathway. Among these, the PI3K-Akt pathway exerts a key role during mammary gland development . Insulinactation . Furthern yields . The MAPn yields . Do et an yields . Wang etn yields . Addition yields . Interesn yields . Insulinynthesis . In goat [INSIG1 . Signal poptosis . Co-exprpoptosis . It is rpoptosis . STAT3 ipoptosis . FibroblTo the best of our knowledge, the present study is the first to detect RNA editing sites in the mammary gland of yaks. We comprehensively analyzed the landscape of RNA editing in the yak mammary gland in the LP and DP. On the basis of these findings, some genes containing the functional edited sites were found to be associated with mammary growth and lactation biology. Most importantly, these findings offer new insights into the mechanism underlying yak mammary gland development at the molecular level and enlarge the yak RNA editing site list.


Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated  in vivo is not fully understood. Here, we identified gain-of-function mutations in the motor domain or CC1 domain that significantly enhance the synaptic vesicle transport. Further biochemical and structural analyses revealed that these mutations could disrupt the CC1/motor mediated autoinhibition. Thus, our work provides a mechanistic explanation for the role of some disease-related mutations in motor hyperactivation.UNC-104/KIF1A is the founding member of the kinesin-3 family. When not transporting cargos, most kinesin-3 motors adopt an autoinhibited conformation, and how the UNC-104/KIF1A motor is activated  C. elegans and is the primary kinesin motor for anterograde axonal transport of synaptic vesicle precursors [Neurons are highly specialized for the processing and transmission of cellular signals. Most proteins necessary for synaptic terminals are transported down axon after synthesis in the cell body. UNC-104/KIF1A was originally identified through genetic screens in ecursors . KIF1A-mecursors . In addiecursors . Given tecursors –9.unc-104 mutants have been identified from genetic screens in C. elegans [UNC-104/KIF1A belongs to the kinesin-3 subfamily  and can  elegans ,23. Thes elegans . Intrigu elegans ,24. HoweC. elegans neurons, while the wild type UNC-104 motor is distributed mainly in cell body, the gain-of-function motors tend to accumulate on particular places of neuronal processes. Once on neuronal processes, the mutant motor possesses similar motility as the wild type, suggesting the disruption of motor-domain-mediated autoinhibition results into more activated motors but not hyper-activated motors in vivo.In addition to the CC2-FHA and NC-CC1 interactions, we now revealed that the intramolecular interaction between the motor domain and the CC1 domain is also important for the autoinhibition of UNC-104/KIF1A motor. Here, the extensive genetic analysis based upon CRISPR/Cas9-mediated genome editing demonstrated that mutations disrupting the CC1/motor interactions result in hyper-activated axonal transport. Structure analysis further showed that the gain-of-function mutation on the motor region could cause an active dimeric conformation. Introducing interface-disrupting mutations into the motor domain or CC1 domain also lead to the gain of UNC-104 function in synaptic vesicle transport. In C. elegans, the cell bodies of DD motor neurons are situated on the ventral side, and they send out ventral-to-dorsal commissures to the dorsal nerve cord. After reaching the dorsal cord, each commissure splits into two branches, one extending anteriorly and the other posteriorly. These two branches form evenly distributed synaptic specializations on the dorsal muscles, which can be visualized with the synaptic vesicle marker GFP::RAB-3 driven by the unc-25 promoter [xd53 mutant  phenotype and display synaptic vesicle accumulation in the cell body region of neuronal cells [xd53 animals are healthy with relatively normal locomotion. Furthermore, the synaptic vesicle clusters in xd53 animals are found on the dorsal cord  worms, we further performed a suppressor screen to isolate secondary site mutations which restored the synaptic vesicle distribution. From this screen, the xd359, xd366, and xd475 mutants were isolated (unc-104(xd53 xd359), unc-104(xd53 xd366) and unc-104(xd53 xd475) worms, the even distribution of GFP::RAB-3 puncta on the dorsal cord was largely reestablished, similar to the wild type (unc-104(xd53 xd359)/unc-104(xd53), unc-104(xd53 xd366)/unc-104(xd53) and unc-104(xd53 xd475)/unc-104(xd53) heterozygote animals, the GFP::RAB-3 distribution pattern resembles that in wild-type animals  mutation site, each suppressor contains a secondary mutation in the coding region of the UNC-104 protein: C184Y in xd359, G421E in xd366 and L410F in xd475 ? One possibility is that the point mutations of xd359(C184Y), xd366(G421E) and xd475(L410F) can correct the protein folding deficiency specifically caused by xd53(lf L640F) and therefore suppress the synaptic vesicle distribution defect only in xd53(lf L640F). Alternatively, the suppressor mutations alone could lead to enhanced synaptic vesicle transport. In this case, the synaptic vesicle trafficking deficiency in other unc-104 loss-of-function mutants may also be suppressed by these dominant suppressors. Thus, we introduced the C184Y, G421E or L410F mutation into another loss-of-function allele, unc-104(xd130). The unc-104(xd130) allele was isolated from the same genetic screen in which unc-104(xd53) was identified. The mutated unc-104 gene in unc-104(xd130) encodes an UNC-104 motor containing a G1092E missense mutation in the C-terminal linker region  animals display an uncoordinated (Unc) locomotion phenotype (xd130(lf G1092E), which restored the even distribution of GFP::RAB-3 puncta on the dorsal cord (xd130(lf G1092E) resulted in a redistribution of the synaptic vesicle clusters to the dorsal cord region. We noticed, however, that the GFP::RAB-3 puncta in unc-104 were not evenly spread along the whole dorsal cord but instead tended to localize around the dorsal branching point of DD neurons (xd130(lf G1092E). We further examined the Unc phenotype and found that the unc-104(xd130) locomotion defect was considerably suppressed by the C184Y, L410F or G421E suppressor mutation  encodes an UNC-104 motor containing a D1497N mutation in the C-terminal PH domain, which may reduce the interaction with cargo [e1265 animals, the synaptic vesicles are retained at the cell bodies [unc-104, unc-104 , and unc-104 worms. Intriguingly, the abnormal synaptic vesicle accumulation defect of e1265(lf D1497N) could not be suppressed by xd359(C184Y), xd366(G421E) or xd475(L410F) (xd359(C184Y), xd366(G421E), and xd475(L410F) suppressors may specifically affect the non-cargo-binding aspect of UNC-104 motors. Alternatively, fewer activated motors in vivo might lead to fewer motors binding to cargo and motors not binding to cargo may undergo degradation [xd359(C184Y), xd366(G421E) or xd475(L410F) on xd53 or xd130 might be due to more active motors which lead to more motors being recruited on the cargo.How do the suppressors repress the mutant phenotype of r region . Comparer region , the GFPd region . Meanwhid region . In addihenotype . Using tsal cord . Introdu neurons . Hence, mutation . e1265 ith cargo . In e126l bodies  S3 Fig)unc-104(xradation . In thisxd53(lf L640F) and xd130(lf G1092E)? To assess the transport function of the UNC-104C184Y, UNC-104L410F and UNC-104G421E motors in vivo, we created xd359(C184Y), xd475(L410F) and xd366(G421E) mutant animals utilizing the CRISPR/Cas9 technique. The xd359(C184Y), xd475(L410F) and xd366(G421E) animals are healthy with normal locomotion. In DD motor neurons, we found that the synaptic vesicle clusters were evenly distributed along the dorsal cord in xd359(C184Y), xd366(G421E) and xd475(L410F) mutant animals  devoid of pre-synaptic structures [unc-104(C184Y) and unc-104(L410F) mutant animals, we found that in addition to the synaptic region, ectopic GFP::RAB-3 puncta appeared in the dorsal distal tip region (in vivo [unc-104(G421E) mutant animals did not display apparent ectopic synaptic vesicles at the distal region of either the dorsal or ventral process. We suspected that the G421E mutation has a relatively weak gain-of-function effect on UNC-104. Indeed, while the synaptic vesicle distribution in xd53(lf L640F) or xd130(lf G1092E) was restored or partially restored by G421E, the G421E mutation alone did not significantly augment the axonal transport in DA9 neurons. The length of the synaptic and asynaptic region is not altered by unc-104(C184Y), unc-104(L410F) or unc-104(G421E) unc-25::Gp region  and the p region , which i , which sxd53(lf L640F) mutants, synaptic vesicles abnormally accumulate in the commissure area of DA9 (xd130(lf G1092E) mutants also display ectopic synaptic vesicles on the commissure area and this phenotype could be suppressed by C184Y, L410F or G421E mutation (xd130(lf G1092E). Based on the average number of GFP::RAB-3 puncta, the xd130(lf G1092E) display a much stronger dendritic accumulation defect than unc-104(C184Y) or unc-104(L410F). Introducing the C184Y, L410F or G421E mutation could significantly reduce the synaptic vesicle clusters in the dendrite region in xd130(lf G1092E)  . Further G1092E) .C184Y and UNC-104L410F compared to wild-type UNC-104 . Compared to the wild type, the anterior movement and the cargo segment run length significantly decreased for UNC-104 lf L640F  function could be restored or even strengthened by C184Y mutation.We further created the UNC-104g region , suggest40F::GFP . In addiy region . In geneC184Y mutant motor is accumulated, we introduced the UNC-104C184Y::GFP into the neuronal marker, specifically labelling PLM neuron [en passant chemical synapses with neurons in the ventral nerve cord. We found that the UNC-104C184Y::GFP generally lacked in the PLM cell body, compared to wild type UNC-104::GFP  on the anterior process of PLN neuron , xd475(L410F) or xd366(G421E)  , further2+) and NC, but not of CC1, possibly due to the intrinsic flexibility of CC1 upon dissociation from the motor domain  mutant using the CRISPR/Cas9 technique. The L428 is located in the CC1 region and corresponding to the L415 site on KIF13B (unc-104(L428A) mutant animals, ectopic synaptic vesicles appear in the distal tip and dendrite region of DA9  mutant gene encodes an UNC-104 motor containing the L170Q mutation and the unc-104(xd407) allele encodes an UNC-104 motor containing the K427A mutation. To test whether xd408(L170Q) and xd407(K427A) behave as gain-of-function alleles of unc-104, we performed the following experiments. Firstly, we examined the synaptic vesicle distribution in DA9 neurons. We found that both the L170Q and K427A mutations lead to ectopic GFP::RAB-3 puncta appearing in the tip and dendritic region of DA9 neurons  and unc-104 mutant animals using CRISPR/Cas9 genome editing. We found that the uneven distribution of DD synaptic vesicle clusters in unc-104(lf 640F) could be rescued by L170Q or K427A mutation (arl-8 and xd408(L170Q) or xd407(K427A). We found that the synaptic vesicle transport defect of arl-8 in DA9 neurons could be efficiently suppressed by xd408(L170Q) or xd407(K427A) mutant (unc-104(lf 640F) mutant DA9 could be suppressed by L170Q or K427A mutation  may also be involved in the direct autoinhibitory contact between the motor domain and CC1 of UNC-104/KIF1A Figs  and 7A.  neurons . We furt animals . Secondlmutation . Thirdly) mutant . Furthermutation . Taken tunc-104. Combined with crystal structure and in vitro ATPase activity measurements, we further revealed that the CC1/motor intramolecular interaction could mediate the autoinhibition of UNC-104/KIF1A. Unlike the transgenic overexpression of UNC-104, we also showed that the endogenous wild type UNC-104 motors are diffusely distributed in cell bodies and neuronal processes. In contrast, gain-of-function motors are generally lacking from cell bodies. Instead, they accumulate on neuronal processes, particularly at the neuronal terminals.Till now, two kinds of intramolecular interactions could mediate UNC104/KIF1A autoinhibition. One is the intramolecular interaction between CC2 and FHA domains, another is NC-CC1 interaction . Here, wC. elegans three decades ago [unc-104. The xd53(L640F) mutation resides in CC2, while the xd130(G1092E) mutation is located in the flexible linker region between CC3 and the C-terminal PH domain. Further modifier screens identified that disrupting the CC1/motor mediated autoinhibition could suppress the reduction of UNC-104 activity caused by different mutations (L640F and G1092E) implying that the uninhibited state of the MD-NC-CC1 region is sufficient to drive UNC-104/KIF1A motor activation. Consistent with this assumption, the structural analysis demonstrated that the T192Y-MD-NC-CC1 mutant adopts an active dimeric conformation  could not be suppressed by gain-of-function mutation C184Y, G421E or L410F. This observation supports the notion that cargo binding is a prerequisite for the cargo transport activity of a UNC-104 motor. Noticeably, two intragenic suppressors of the mutation in the PH domain have been identified. It appears that while loss of cargo binding reduces motor levels, these two suppressors could restore the UNC-104 level and cargo binding [Genetic screens have been widely used to explore the molecular, cellular and developmental mechanisms underlying various biological processes. In fact, UNC-104/KIF1A, the axonal transport kinesin for synaptic vesicles and the founding member of the kinesin-3 family, was originally identified through a locomotion behavior screen in ormation . The los binding . It willC184Y tends to accumulate on neuron processes. In PLM neurons, the UNC-104C184Y motor is enriched at the terminal region of the anterior branch but not the posterior end of PLM. Our previous studies demonstrated that actin is concentrated at the terminal region of the anterior PLM process, in which the PLM forms gap junctions with interneuron BDU [C. elegans neurons [C184Y::GFP dots does not resemble the distribution pattern of gap junction connections. For PLN neuron, it is completely unclear whether any special cytoskeleton arrangement exists in the two places on which the UNC-104C184Y::GFP is accumulated. Hence, why UNC-104C184Y motor specifically accumulates on above positions and whether the abnormal accumulation leads to any functional consequence of C. elegans neurons remain to be determined. Considering the synaptic vesicle transport is enhanced in unc-104(C184Y) mutants and the cell-body-distribution of UNC-104C184Y motors is greatly reduced, it is a reasonable proposal that the C184Y mutation could lead to more motors in an activated state.The gain-of-function motor UNC-104uron BDU . Gap jun neurons . Howeverunc-104 that caused hyperactive axonal transport of synaptic vesicles  mutant animals display typical gain-of-function features of unc-104 and subcellular localization of UNC-104(L428A)::GFP resembles C184Y::GFP. Furthermore, when two new mutations in the UNC-104 motor domain or CC1 predicted to disrupt the inter-domain autoinhibitory interface were introduced into UNC-104 motor, the point mutations resulted in gain of UNC-104 function and hyperactive axonal transport in vivo , xd359(C184Y), xd475(L410F), xd366(G421E), xd407(K427A), xd496(L428A), xd53(lfL640F), xd130(lfG1092E), e1265(lf D1497N), xd53 xd408, xd53 xd359, xd53 xd475 , xd53 xd366 , xd53 xd407,xd130 xd359 , xd130 xd475, xd130 xd366 , xd500 xd359, xd501 xd475, xd502 xd366, xdKi3(unc-104::gfp knock-in), xdKi6(C184Y::gfp knock-in), xdKi60(L428A::gfp knock-in),xdKi4(lf L640F::gfp knock-in), and xdKi61. Other strains are: xdIs164(Punc-25::GFP::RAB-3),xdEx968 (Pmec-7::mCherry) and xdEx2678 (Plad-2::mCherry). wyIs85(Pitr-1::GFP::RAB-3) was provided by Kang Shen (Stanford University). The unc-104 (xd53) and unc-104 (xd130) worms were isolated from xdIs164 worms treated with EMS. Thexd53 xd359, xd53 xd475 , xd53 xd366  worms were obtained from an EMS-based genetic screen usingunc-104(xd53);xdIs64 strain. Briefly, the distribution of synaptic vesicles in F2 progeny was examined under a fluorescence microscope, and suppressor mutant animals were recovered to produce progeny. A total of 7,200 mutagenized haploid genomes were screened and total eight suppressors were isolated from this screen.escribed . Mutantsunc-104cDNAs were amplified by PCR and cloned into dpSM vectors . psc325 vector contains the unc-25 promoter. Transgenic animals were made by standard microinjection procedures. In general, plasmid DNAs of interest were used at 1–50 ng/μl, and the co-injection marker was Pmyo-2::RFP or Podr-1::RFP or Pord-1::GFP. All transgenic strains were crossed to N2 wild type 4 times before use. The CRISPR/Cas9-mediated genome editing strains were generated by SunyBiotech and were verified with DNA sequencing. All strains were outcrossed with N2 for 4 times before use. The detailed information is included in Animals were mounted on 2.5% agar pads in M9 buffer containing 1% 1-phenoxy-2-propanoland. Fluorescence photographs were taken using a Leica SP8 confocal microscope unless specifically indicated. The GFP::RAB-3 fluorescence images of DD/VD neurons were taken at the young adult stage. The GFP::RAB-3 fluorescence images of the DA9 neuron were taken at the first day of adulthood. Straightened dorsal cord images were obtained using ImageJ software. The lengths of the synaptic region and the asynaptic region were measured by ImageJ. For each genotype, more than 20 worms were imaged.Time-lapse images of GFP::RAB-3 were taken at the L4 stage, using an inverted Zeiss Axion Observer Z1 microscope equipped with a Plan-Apochromat 63x/1.4 objective, an Andor EM-CCD digital camera, and a Yokogawa CSU-X1 spinning disk system. 201 frames were taken for each movie with a speed of 6 frames per second and an exposure time of 160 ms. For movie acquisition, worms were mounted on 2.5% agar pads and anesthetized with 1-phenoxy-2-propanol. Movies were analyzed using ImageJ. The “register” plugin was used for movie correction. Subsequently, “segmented line” was used to select the region for kymograph analysis. Finally, kymographs were constructed with the “kymograph” plugin. For each genotype, 30 worms were imaged.Each contiguous line with a constant slope was scored as a single transportevent and its run length  were calculated. Run length is defined as a singlemotility occurrence typically right after a pause or a reversal and,vice versa, ends when the motor is again pausing or reversing.The FRAP experiments were performed on the young adult animals using a laser scanning confocal microscope  equipped with a Plan-Apochromat 63x/1.4 NA oil immersion. For bleaching, worms were mounted on 2.5% agar pads and anesthetized with 1-phenoxy-2-propanol. Photobleaching, image acquisition, and image analysis were performed on ZEN system. Image acquisition was conducted for regions with 792 x 792 pixels (0.09μm/pixel) using 488-nm semiconductor lasers at 10% power with the pinhole diameter 56μm. Prior to the bleaching, 3 frames of images were acquired. Photobleaching was performed on a rectangle region with size 55 x 22 pixels using 488-nm lasers at 20% power for 250ms. After photobleaching, images were collected till no further recovery was detected. The fluorescence recovery of the bleached region was calculated as follows. Firstly, average fluorescence intensities within the bleached region and non-bleached regions  were measured. Second, the lowest intensity value (immediately after bleaching) within the photo bleaching region was subtracted from all measurements. The average intensities of the 3 measurements that precede the bleaching were then determined, establishing a pre-bleach value; all intensities were normalized by dividing by that value. The halftime of recovery is the time point where the fluorescence recovered to half of the maximum recovery.post hoc test (the test used is stated in each figure legend). * P<0.05; ** P<0.01; *** P<0.001.To compare multiple groups, one-way ANOVA was used with an appropriate multiple comparisons Worms were assessed for Unc phenotypes and locomotion defects by examining their motility on NGM plates. Young adult worms were transferred to new plates and movement was recorded under a dissection microscope (Nikon) equipped with a HQ CCD camera (Aoka).For velocity assay, movements were recorded at 1–2 frames/s. The obtained movements were tracked using a custom programmed ImageJ’s “multitracker” plugin.Young adult worms were cryofixed on a Leica Microsystems HPM 100 and frozen in liquid nitrogen. After high-pressure freezing, the samples were washed 4 times in acetone and stained with 1% uranyl acetate for 1 hr. The infiltration was performed with increasing concentrations of Embed 812. Finally, samples were embedded in fresh resin at 60°C for 3 days. Thin sections (60 nm) were produced with a diamond knife (Diatome) on an ultramicrotome . Serial sections were pictured with a JEM-1400 electron microscope at 80 kV. Pictures were recorded using a Gatan832 4k × 2.7k CCD camera.Worms were collected and washed in M9 buffer. Lysis buffer  was added and worms were disrupted with a Dounce homogenizer  on ice for 15 min. Debris was removed by centrifuging at 12000 rpm for 15 minutes at 4°C. For western blotting, protein samples were boiled and separated in an 8% SDS-PAGE gel. After transfer, the nitrocellulose membranes  were incubated with 5% dry milk and probed with an anti-GFP antibody . The signals were visualized with a Supersignal West Pico Substrate kit (Pierce). The stained bands were analyzed by ImageJ Software. Graphical data are presented as mean ± SEM. n = 3 independent experiments.6-tag. Point mutations were introduced using the standard PCR-based mutagenesis method and confirmed by DNA sequencing. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) host cells at 16°C. The His6-tagged fusion proteins were purified by Ni2+-Sepharose 6 Fast Flow  affinity chromatography followed by size-exclusion chromatography  in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT. For analytical gel-filtration analysis, protein samples were concentrated to ~2.5 mg/ml and loaded onto Superdex-200 10/300 GL columns .DNA sequences encoding rat KIF13B MD-NC-CC1 and various mutants were cloned individually into the pET32a vector with a carboxyl HisMeasurements of the microtubule-stimulated ATPase activities of KIF13B MD-NC-CC1 and various mutants were performed using the HTS Kinesin ATPase Endpoint Assay Biochem Kit . Briefly, all the measurements were based on the malachite green phosphate assay to probe inorganic phosphate generated during the reaction. A standard curve of phosphate was made to estimate the amount of phosphate generated. The basal ATPase activities (without microtubules) of these KIF13B fragments were also measured using the same method. Each protein sample had three replicates and each measurement was repeated at least three times independently. The kinesin-1 heavy chain (KHC) supplied in the Kit was used as the control.2Fo-Fc and Fo-Fc electron density maps using COOT [Crystals of the KIF13B T192Y-MD-NC-CC1 mutant (~25 mg/ml) were obtained in 0.1 M Bis-Tris propane, pH 8.2, 30% (w/v) PEG 20000 using the sitting drop vapor diffusion method at 16°C. Before being flash-frozen in liquid nitrogen, crystals were soaked in the mother liquor supplemented with 15% (v/v) ethylene glycol for cryo-protection. The diffraction datasets were collected at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility with a wavelength of 0.989 Å at 100K . Dataseting COOT . The string COOT . The staS1 Figwt) and unc-104(xd53). White arrows indicate DD neurons. White brackets indicate regions lacking GFP::RAB-3 puncta. The schematic drawings on the right show the synaptic vesicle distribution in DD neurons. (C and D) EM images of dorsal synapses in wt and unc-104(xd53). M, muscle. White arrows indicate active zones. Scale bar represents200 nm. (E-H) The distribution of Punc-25::GFP::RAB-3 (green) puncta in wt, unc-104, unc-104, and unc-104 worms. Scale bar represents 50 μm.(A and B) The distribution of GFP::RAB-3 puncta (green) in wild type ((TIF)Click here for additional data file.S2 Figunc-104(lfG1092E), unc-104, unc-104, and unc-104 worms. Dashed circles indicate the spots on which 15 worms of each genotype were placed. Scale barrepresents1 mm. (E) Quantification of the velocity in various genotypes. ***P<0.001, one-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 15 worms for each genotype.(A-D) Snapshots were taken at 10 s, 5 min and 20 min of (TIF)Click here for additional data file.S3 Figxd359(C184Y), xd475(L410F), xd366(G421E) and e1265(lf D1497N). xd359, xd475 and xd366 are indicated. (B) The even distribution of GFP::RAB-3 puncta on the dorsal cord is not restored in unc-104, unc-104 and unc-104 animals.(A) Schematic drawing of the domain organization of UNC-104 motor protein. The mutation sites of (TIF)Click here for additional data file.S4 Figunc-25 in xd359 (C184Y) (A), xd475 (L410F) (B) and xd366 (G421E) (C). Scale bar represents 50 μm. (D) The schematic drawing shows the synaptic vesicle distribution of DD neurons. (E-F) The abnormal synaptic accumulation defect in unc-104(lf L640F) could be suppressed by C184Y (G) or G421E (H) mutation on UNC-104. (I)Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. (J) Quantification of the length of the asynaptic region. **P<0.01; NS, not significant. One-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 20 worms for each genotype.(A-C) The distribution of GFP::RAB-3 puncta (green) driven by P(TIF)Click here for additional data file.S5 FigC184Y motors. (B) Quantification of the expression level of the motor proteins in (A). Mean ± SEM, two-tailed paired Student’s t test. NS, not significant. (C) Quantification of the percentage of the worms with GFP accumulation on the anterior tip region of PLM neuron. Mean ± SEM, two-tailed unpaired Student’s t test. Total 300 worms were examined for each genotype. N = 6. (D) Quantification of the percentage of the worms with normal PLM morphology. Mean ± SEM, two-tailed unpaired Student’s t test. About 130 worms were examined. N = 3. (E) Quantification of the percentage of the worms with normal synapse formation. Mean ± SEM, two-tailed unpaired Student’s t test. 250 worms were examined. N = 5. ***P<0.001; NS, not significant.(A) Western blot of worm lysates probed with a GFP antibody to detect the expression level of wild-type and UNC-104(TIF)Click here for additional data file.S6 Fig2+ (contoured at 1.5σ level).(A) Analytical gel-filtration analysis of wild type and various mutants of the MD-NC-CC1 region of KIF13B . (B) Microtubule-stimulated ATPase activity of the UNC-104 MD-NC-CC1 fragment containing the C184Y, L410F, G421E, or L428A mutation. Bars represent Mean ± SD. n = 4 independent experiments, * P<0.05, one-way ANOVA with LSD test. (C) Structure of the T192Y-MD-CC1 mutant fragment of KIF13B. The inset shows the omit electron-density maps of ADP and Mg(TIF)Click here for additional data file.S7 Figarl-8, arl-8;unc-104(C184Y),arl-8;unc-104(L410F)and arl-8;unc-104(G421E) worms. The synaptic region is shown on the top. Scale bar represents 25 μm. (F) Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. (G) Quantification of the length of the asynaptic region. ***P<0.001, one-way ANOVA with Tukey test. Mean ± SEM, N> = 20 worms for each genotype.(A-E) The GFP::RAB-3 puncta distribution in wild type, (TIF)Click here for additional data file.S8 Figwt) unc-104(L428A). Time and length are on the y axis and x axis, respectively. (B) The number of stable GFP::RAB-3 particles in a 1-μm section within 1 min. (C) The number of anterograde transport events. (D) The ratio of anterograde transport events to retrograde transport events. **P<0.01, ***P<0.001. Mean ± SEM, two-tailed paired Student’s t test. N = 30 worms for each genotype. (E-H) The even distribution of GFP::RAB-3 (driven by Punc-25 promotor) puncta on the dorsal cord is restored in unc-104 and unc-104 animals. (I-L) The synaptic vesicle transport defect of arl-8 mutants could be suppressed L170Q or K427A mutation (M) Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. The synaptic region is shown on the top. Scale bar represents 25 μm. (N) Quantification of the length of the asynaptic region. ***P<0.001, one-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 20 worms for each genotype.(A) Representative kymograph images showing transport events in wild type ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Stress granules (SGs) are phase-separated, membraneless, cytoplasmic ribonucleoprotein (RNP) assemblies whose primary function is to promote cell survival by condensing translationally stalled mRNAs, ribosomal components, translation initiation factors, and RNA-binding proteins (RBPs). While the protein composition and the function of proteins in the compartmentalization and the dynamics of assembly and disassembly of SGs has been a matter of study for several years, the role of RNA in these structures had remained largely unknown. RNA species are, however, not passive members of RNA granules in that RNA by itself can form homo and heterotypic interactions with other RNA molecules leading to phase separation and nucleation of RNA granules. RNA can also function as molecular scaffolds recruiting multivalent RBPs and their interactors to form higher-order structures. With the development of SG purification techniques coupled to RNA-seq, the transcriptomic landscape of SGs is becoming increasingly understood, revealing the enormous potential of RNA to guide the assembly and disassembly of these transient organelles. SGs are not only formed under acute stress conditions but also in response to different diseases such as viral infections, cancer, and neurodegeneration. Importantly, these granules are increasingly being recognized as potential precursors of pathological aggregates in neurodegenerative diseases. In this review, we examine the current evidence in support of RNA playing a significant role in the formation of SGs and explore the concept of SGs as therapeutic targets. Stress granules (SGs) have been described as a triage for mRNA during cellular stress where they either store translationally silent mRNA, transfer mRNA transcripts to processing bodies (p-bodies) where they will be degraded, or transfer mRNA back into polysomes for translation . This SGCellular stress can induce the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), which is generally considered the trigger that induces SG assembly. However, SG formation can be independent of eIF2α phosphorylation and triggered by eukaryotic translation initiation factor 4A (eIF4A) . In eithThe coalescence of RBPs and mRNA molecules into SGs creates a ribonucleoprotein (RNP) granule. This non-membrane bound organelle contains two subcompartments, the shell and the core. Cores are highly concentrated areas of mRNA and protein, which are surrounded by a less concentrated area, known as the shell, which is believed to be more dynamic . Little The assembly of proteins during periods of stress results in the liquid–liquid phase separation (LLPS) of SGs that is primarily driven by weak electrostatic, hydrophobic, and homo- and heterotypic protein–protein interactions between RBPs that contain intrinsically disordered domains . While pThese liquid-like structures are highly dynamic and continuously undergo adenosine triphosphate (ATP)-dependent remodeling . For exaIn vitro oxidative stress induced with sodium arsenite results in the formation of SGs within 30 min, while double-stranded DNA breaks induced by UV exposure in vitro results in a much slower formation of SGs spanning upward for 18 h  within 3 h as SG-related proteins  peak in expression  or puromycin (a polysome destabilizer) results in decreased or increased SG formation, respectively  and Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) are two RBPs that are necessary components for SG formation . The aggOnce nucleation occurs, this allows for the recruitment of other proteins with a variety of functions, creating a complex composition that allows SGs to dynamically exchange RNA and protein molecules. These additional proteins associated with SG include translation initiation factors, RBPs, kinases, phosphatases, ATPases, guanosine triphosphatases (GTPases), methyltransferases, ribosyltransferases, glucosyltransferases, DNA/RNA helicases, and ubiquitin-modification enzymes . The speImportin β1 (Impβ1) mRNA, but dissociates Neuritin 1 (Nrn1) allowing for decreased and increased translation of these two transcripts, respectively, which is believed to promote rapid regenerative processes  is important for functions related to synaptic plasticity or axonal regeneration . In yeasNuclear SGs, termed nuclear stress bodies (nSB), have also been described as a component of the heat shock response. The main component of the nSB is the transcription factor heat shock factor 1 (HSF1), which nucleates in the SAT III region of DNA and transcribes a series of non-coding RNAs . TAR DNAStress granules are critical determinates for RNA-mediated gene expression in which the composition of these molecules is driven by specific cell stresses. They can act as an intermediate between polysomes and p-bodies to regulate RNA translation and degradation, respectively. Overall, this regulation of mRNA metabolism/expression by SGs allows cells to maintain homeostasis in response to a stress, or when the stress cannot be overcome, induce apoptotic pathways. In recent years, RNA has become increasingly implicated in SG assembly and disassembly. Studying RNA composition of SGs and its function is of outmost importance not only because RNA is the major component of SGs but also because as we are going to see later, RNA itself is able to phase separate in the absence of proteins and to function as efficient scaffolds for protein complexes. The more we understand about RNA molecules in SGs, the more we are going to increase our knowledge about SG dynamics, and the closer we are going to be to finely regulate these transient organelles and to develop therapeutic tools for different types of diseases.Several RBPs have been observed to drive the formation and dynamics of SGs. Overall, protein drivers contain intrinsically disordered regions (IDRs) and/or govern low-affinity multivalent interactions . The funRNA is a highly flexible and dynamic molecule whose folding is more promiscuous than protein folding due to the simpler alphabet and base-pairing rules. An RNA molecule spontaneously adopts a vast number of conformations that together are called ensembles , some ofcis) and heterotypic (trans) RNA–RNA . The stress sensors are kinases HRI , PKR , PERK , and GCN2 ; all of which phosphorylate eIF2α . This evinto SGs . For man2+ and H+ do not have the chaperone activity necessary to drive secondary structure transitions over the associated large-energy barriers. This is the reason that they act to direct RNA to different folding pathways during RNA co-transcriptional folding  in the chromosome 9 open reading frame 72 (C9orf72) gene in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It has been shown that GC repeats in a G-quadruplex conformation originate in RNA sol–gel transitions in vitro and in cells, without requiring proteins, in a repeat length and structure-dependent manner and at a similar repeat number as in nucleotide repeat expansion disorders. RNA foci are formed by phase separation of the repeat-containing RNA in cells and can be dissolved by agents that disrupt RNA gelation  thermodynamic RNA condensers, proteins that reduce ΔG of RNP granulation through RNA binding; (c) kinetic RNA decondensers, proteins that reduce activation energy barriers and decrease the valency of RNAs to promote the dissociation of cis or trans RNA interactions and accelerate RNA refolding; (d) thermodynamic RNA decondensers proteins that bind RNA with high affinity to restrict the sites or conformations that are available for trans RNA–RNA interactions; and (e) client/unknown proteins. According to this model, most SG proteins would assist to control RNA condensation through ATP-dependent processes  and formin (BNI1) transcripts in the cytosol of large cells in fungi  proteins that include paraspeckle component 1 (PSPC1), non-POU domain-containing octamer-binding protein (P54NRB/NONO), and splicing factor proline and glutamine rich (SFPQ/PSF). NEAT1, unlike DBHS proteins that are also necessary for paraspeckle formation, is a limiting factor in the formation of these granules. The two transcript isoforms of NEAT1, NEAT1_1  and 1_2  are necessary to maintain paraspeckle integrity (Perhaps the most well-known example of RNAs as nucleating factors of RNP granules is the long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the formation of stress-inducible nuclear bodies called paraspeckles . There antegrity .in vivo and are necessary for the assembly of SGs  lengths form extended RNA–RNA networks y of SGs   sites as a mechanism of both SG assembly and mRNA stability control . Long 3′D. melanogaster is the 3′UTR (A comparison of the different RNP granule isolation techniques performed by he 3′UTR .Using tail-end displacement sequencing (TED-Seq) for transcriptome-wide profiling of poly(A) lengths, Cumulative evidence has shown that the cellular response to stress is finely regulated through post-translational modification (PTM) of proteins. The specific function of PTMs in SG dynamics has been extensively studied, leading to the proposal that alterations in physicochemical properties of modified amino acids regulate SGs, weakening or enhancing the multivalent interactions between macromolecules, or recruiting or excluding macromolecules from the granules .Recently, the role of RNA chemical modifications in SG formation dynamics has started to be revealed. RNA modifications are critical at different steps of the RNA metabolism, from transcription and splicing, to stability, translation, and RNA function. The mRNA epitranscriptome contains methyl, hydroxymethyl, acetyl, formyl, as well as the nucleoside isomers, pseudouridine and inosine. RNA molecules undergo a variety of reversible and irreversible modifications through a highly regulated process in which different enzymes participate. Writer enzymes transfer specific chemical groups to the RNA molecule, eraser enzymes remove them, and reader enzymes recognize modified nucleotides .6A). This modification has been found in tRNAs and rRNAs and is enriched around the stop codon of mRNAs and in 3′ UTRs. Its role is to regulate mRNA stability, localization, splicing, and translation  and insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1-3) mediate methylation effects . This highly conserved modification not only adds a methyl group but also a positive charge to the RNA at the Watson–Crick interface, blocking RNA base pairing. Importantly, m1A increases in mRNAs in response to stress. m1A is also a well-known modification in rRNAs and tRNAs, playing roles in maintaining their biological functions. Also, it has been shown to be enriched around the start codon of mammalian transcripts where it promotes translation in methylated mRNAs and has been found in certain lncRNAs , cell cycle-associated protein 1 (CAPRIN1), and RNA-binding motif protein 42 (RBM42), all proteins of SGs  and a high tendency to phase separate, is capable of forming or regulating RNA granules through interactions with host SG-RNAs and proteins . A serieOn the other side, many viruses induce the formation of intracellular compartments in the host cell called viral factories, structures that concentrate nucleic acids and viral proteins, as well as some specific cellular factors. Viral factories facilitate viral replication and assembly and protect the viral genome from the cell defense mechanisms. Interestingly, some viruses form nuclear or cytoplasmic membraneless viral factories that have liquid organelle properties . An examFUS gene are associated to ALS RNA that mimics viral infection, polyinosinic–polycytidylic acid [poly(I:C)], induces persistent formation of mutant FUS/TLS SGs. FUS/TLS is an RNA-/DNA-binding protein that forms pathological inclusions in ALS, and mutations in d to ALS . Poly and V-Ets avian erythroblastosis virus E26 oncogene homolog (ETS2), and several rearrangements between domains of different transcription factors and the RNA-binding domain of EWSR1 or the N-terminal domain of EWSR1 or TAF15 have been reported in cancer . These and other stressors could be lethal for tumor cells unless they rapidly adapt through stress-resilient clones . EmergenThird, there is evidence that interference with the dynamic of SGs negatively impacts on cancer. Knocking-down G3BP1 reduces SG assembly in cancer cells treated with bortezomib (BZM), a proteasome inhibitor used for a range of hematological tumors, potentiating chemotherapeutic-induced cancer cell death . FurtherAltogether, these data suggest that inhibiting SG assembly and changing the stress conditions of the tumor can influence tumor progression and sensitize cancer cells to chemotherapeutic agents, representing a promising strategy for cancer treatment. It also suggests that because drugs selectively partition into membraneless organelles, cells can develop drug resistance through a mechanism that involves condensates . This shRNA-binding proteins that localize in SGs such as TIA-1, TDP-43, and G3BP1 have been observed in pathological aggregates of neurodegenerative diseases like Alzheimer’s, Huntington’s and ALS. These findings suggest that SG dysregulation might have a role in protein aggregate formation in neurodegeneration .4C2 repeat expansions and the most common genetic cause of ALS and FTD, has been shown to induce the formation of SGs that colocalize with aggregated poly(GR)  .In vitro experiments have shown that liquid-like droplets of SG proteins, such as FUS/TLS and TDP-43, convert into an aggregated state in time and that this conversion is accelerated by disease-associated mutations (in vivo studies using a light-inducible SG system based on optogenetic multimerization of G3BP1 allowed to demonstrate that persistent formation of SGs is cytotoxic. These SGs evolve in time to neuronal pathological inclusions characteristic of ALS/FTD (FUS/TLS knock-in ALS mice model transduced with TIA-1-EGFP have shown intense TIA-1-EGFP-positive granules formed in the cortex neurons in hours but cleared weeks after stress challenge. Neurons showing severe granule misprocessing die days after stress challenge, demonstrating that SG dysregulation is pathogenic in ALS (C9orf72 ALS mice model have shown that G4C2 repeats induce the formation of aberrant SGs and phosphoTDP-43 pathological inclusions (utations . Importa ALS/FTD . The arr ALS/FTD . Similarc in ALS . Finallyclusions .A general consequence of the formation of pathological SGs is the alteration of the normal function of these granules and the sequestration of specific RBPs, compromising the function of these proteins and altering several cellular processes . For exaMotor neurons might be particularly vulnerable to SG dysregulation and pathological aggregate formation . The reain vitro and in vivo studies are quickly generating extremely valuable knowledge that will be the foundation to develop therapeutic approaches to prevent the transition from dynamic SGs to irreversible inclusions in these devastating diseases.Even though other lines of investigation suggest that there could be more than one mechanism for pathological aggregate formation in neurodegeneration , today, The emergence of the role of the RNA in RNP granule architecture is creating a more complete picture of the functioning of these dynamic organelles. Now, we know that not only specific proteins are necessary for the assembly of SGs but also RNA molecules with special characteristics. RNA is able to phase separate in the absence of proteins and work as efficient scaffolds for protein complexes. Moreover, the RNA structure drives interaction with proteins. In this review, we propose that under stress conditions, RNA and/or enzymes that chemically modify RNA could bind metabolites and ions, triggering changes in the structure and local charges of RNA molecules that prompt RNA–RNA interactions, RNA phase separation, and SG nucleation, contributing to the composition and organization of these RNP assembles .Our knowledge to this point is still limited, and there are still important questions to be answered. How are RNAs selected to phase separate and nucleate SGs if there is no apparent specificity in the process and any RNA could potentially partitionate into these organelles? If there are RNA destination signals to SGs, at which structural levels are they encoded, and do they depend on stress sensor properties of the RNA molecule? Are RNA molecules recruited into SGs in a stepwise process? Finally, are RNAs subcompartmentalized in different domains and contain specific functions inside SGs? The elucidation of these and other interesting questions will allow us to move several steps forward in the understanding of the function of RNA in these dynamic structures.in vitro reconstitution of condensates and cell culture lines have been extremely useful in understanding the dynamic of RNP granules, these systems also have well-recognized limitations (In vivo experiments in yeast and mice are starting to provide invaluable information regarding the physiological relevance of in vitro findings and their connection with pathological stages (S. cerevisiae has determined that during glucose deprivation, proteins are sequestered into SG in a process triggered by ATP exhaustion. In this study, Hsp104 chaperone ATP hydrolysis activity was demonstrated to determine aggregate dissolution and protein aggregate steady-state size (Even though itations . In vivol stages . Recentlate size .in vivo studies of the dynamic of SG formation, we anticipate that advances in the RNA structural field are going to open enormous possibilities to understand different RNP granules and discover new roles for RNA molecules. Recently, two techniques have been developed to study RNA structure. Single-particle cryo-electron microscopy has been used together with M2-seq biochemical analysis and computer modeling to determine 3D models of 11 different RNA molecules (Together with olecules , and dimolecules . AlthougRNA molecules have been increasingly recognized as potential therapeutic targets for different diseases, not only because by targeting mRNAs, functions of proteins that are very difficult to drug or undruggable might be modulated, but also because the vast majority of the human genome encodes for non-coding RNAs . InvestiDC-M, ZH, and MS conceptualized the study. DC-M prepared the original draft. DC-M, ZH, CD, and MS wrote, reviewed, and edited the manuscript. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We analyzed the linear refractive indices, n(λ) of tRNA TSFs on silicon substrates by using an ellipsometer in the 400 to 900 nm spectral range to find a linear correlation with the tRNA concentration in the aqueous solution. The thermo-optic coefficient (dn/dT) of the films was also measured to be in a range −4.21 × 10−4 to −5.81 × 10−4 °C−1 at 40 to 90 °C. We furthermore characterized nonlinear refractive index and nonlinear absorption of tRNA TSFs on quartz using a Z-scan method with a femtosecond laser at λ = 795 nm, which showed high potential as an efficient nonlinear optical material in the IR spectral range.We successfully obtained transfer ribonucleic acid (tRNA) thin solid films (TSFs) using an aqueous solution precursor in an optimized deposition process. By varying the concentration of RNA and deposition process parameters, uniform solid layers of solid RNA with a thickness of 30 to 46 nm were fabricated consistently. Linear absorptions of RNA TSFs on quartz substrates were experimentally investigated in a wide spectral range covering UV–VIS–NIR to find high transparency for  Optical measurements of one of the vital biological molecules (RNA) in the human body. For instance, DNA-TSF has been used as an electron blocking layer in organic light-emitting diodes (OLEDs),8,9 hydrogen-blocking layer in hybrid thin-film transistors (TFTs),10,11 a cladding layer in an optical waveguide modulator,12 a sensing medium in optical sensors,13,14 a nonlinear saturable absorber in femtosecond lasers,3 and a radiation absorbing layer in solar cells,15,16 to name a few. In contrast to DNA, ribonucleic acid (RNA) research has been primarily confined to traditional biochemical and pharmaceutical usages.17–19 In recent years, RNA-based vaccines have become a preventive necessity to curb the global pandemic.20–28 While DNA is a double strand, RNA forms a single strand of the nucleobase arrays and is categorized into messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).20 RNA shares its constituents, adenine (A), guanine (G), cytosine (C), with DNA. Uracil (U) in RNA replaces thymine (T) in DNA.Deoxyribose nucleic acid (DNA) has recently shown prominent potential as a highly functional electro-optic material.25,29 which makes RNA TSF advantageous in an environment exposed to radiation containing UV spectral components. In RNA aqueous solution, we experimentally found that its viscosity was significantly lower than DNA solutions for the same concentration level. RNA aqueous solution precursors provided a superb thin film fabrication capability, especially in the spin coating process, compared to DNA precursors. Despite nearly the same constituents as DNA, RNA has not been thoroughly studied as an optoelectronic substance, and especially RNA TSFs for photonic applications have not been reported to the best knowledge of the authors.RNA compounds are more resistant against UV radiation than DNA counterparts,6,10 for the first time. We further analyzed their linear optical properties in terms of absorption, α, in the UV–VIS–NIR spectral range, linear refractive index, n(λ), in the visible to NIR from 400 to 900 nm, and thermo-optic coefficient, dn/dT, in the temperature range from 40 to 90 °C. Utilizing these linear optical properties, we furthermore investigated the nonlinear refractive index, n2, and the nonlinear absorption, β, using a femtosecond laser Z-scan technique,30 revealing the high potential of tRNA TSF in nonlinear optical applications.In this study, we report a systematic process to fabricate tRNA TSFs with various thicknesses on both silicon (Si) and silica substrates using a surfactant-free aqueous solution of tRNA from wheat germ,A schematic diagram for our study is presented in . We deven, α, and dn/dT) and nonlinear optical characteristics (n2 and β). It was found that the linear optical characteristics were dependent upon the concentration of tRNA prior to thin film deposition. Experimental details are discussed in the following sections.For these samples, we experimentally investigated the linear optical properties  with high purity.icted in .2) drying, oxygen plasma surface treatment. See  in the spectra range of 200–900 nm. The results are summarized in . The tRN34–39 In , the pea3.2λmax ∼ 260 nm, the same as in the aqueous solution in , and the results are summarized in . The peaution in . The simution in , and  relations.7n(λ) in (α(λ) in  in , and α(λ(α(λ) in  for the (α(λ) in  is suffi(α(λ) in , we plotkness in  to find 3.4n/dT. By mounting tRNA TSF on a Peltier thermoelectric device installed at an ellipsometer, we were able to measure its n(λ) in the temperature range from 40 to 90 °C, and the results are summarized in /α is the effective thickness of the sample, calculated with the linear absorption coefficient α = 0.01588 μm−1 and the actual sample thickness L. The nonlinear refractive index n2 was estimated by eqn λ is the laser wavelength and ΔΦ is the nonlinear phase change.Then, the nonlinear absorption β ∼ −1.12 × 10−10 m W−1 in tRNA film, where the negative sign indicates the saturable absorption capability. We also estimated n2 ∼ 1.00 × 10−12 cm2 W−1, where the positive sign was due to the significant difference between the laser energy and the bandgap of tRNA (>∼4.7 eV).56 We compared both β and n2 of tRNA with those of DNA and DNA-CTMA films in  in a range from −4.21 to −5.81 × 10−4 °C−1 for 2.0 wt% tRNA TSF, which suggests further applications to temperature sensing and thermo-optic devices. Nonlinear optical properties of tRNA TSFS were characterized by the Z-scan method, and we estimated a large negative nonlinear absorption of β ∼ 1.12 × 10−10 m W−1 and positive nonlinear refractive index n2 ∼ 1.00 × 10−12 cm2 W−1 at λ = 795 nm, which were comparable or larger than those of DNA films. The large magnitude of β and n2 strongly indicates tRNA TSFs can open new nonlinear optical applications.We successfully developed a new method to fabricate surfactant-free tRNA thin solid films (TSFs) by using tRNA aqueous solution precursors and we precisely controlled their optical characteristics. Both linear and nonlinear optical properties of tRNA TSFs showed their high potential in all-RNA photonic device applications. By measuring the absorbance, we could confirm that tRNA were successfully imbedded in solid films without structural changes. The refractive index at There are no conflicts to declare.