Tomato torrado virus (ToTV) was identified on tomato crops in the Limpopo province of South Africa and a first report of the disease was published. In this follow-up study, the full genome sequence of a tomato-infecting isolate of ToTV from South Africa was elucidated. High-throughput sequencing was used to generate the full genome of ToTV infecting tomato crops in South Africa. The longest contig obtained for the RNA-1 and RNA-2 genome of ToTV was comprised of 7420 and 5381 nucleotides (nt), respectively. Blast analysis of the RNA-1 sequence of ToTV from South Africa (ToT-186) matched 99% to a Spanish and Polish isolate; the RNA-2 segment of ToTV from South Africa (ToT-186) matched 99% to ToTV isolates from Italy and Poland, respectively. The information presented in this study will go a long way towards better understanding the emergence and spread of ToTV and devising sustainable management of ToTV diseases.Emerging pests and diseases are a major threat to food production worldwide. In a recent survey,  Tomato torrado virus”. Although this newly discovered species was shown to display several characteristics similar to the Sequivirus, Sadwavirus, and Cheravirus genera in the Sequiviridae family, sequence characteristics distinguished Tomato torrado virus (ToTV) as a member of a new plant virus genus [Emerging pests and disease have destroyed agricultural crops all over the world. More than fifteen years ago, local farmers in the city of Murcia (located in the southeastern parts of Spain) observed severe necrosis on the leaves and fruit of tomato crops. These symptoms later became known as “torrado” disease which was coined by local Spanish farmers describing the “burnt-like” effect of the disease in tomato fields. A subsequent study by Verbeek et al.  providedus genus .According to Sanfacon et al. . ToTV isTorradoviruses have a bipartite genome composed of an RNA-1 (7.8 kb) and an RNA-2 (5.4 kb) segment. Each genomic segment has a VPg linked to its 5′ end and a 3′ poly (A) tract. RNA-1 and RNA-2 are translated into two polyproteins, which are then processed into functional proteins. RNA-1 encodes proteins  that are necessary for replication while RNA-2 encodes three coat proteins and proteins involved in virus movement. It is further demonstrated that the genome of torradoviruses consists of an exclusive second open reading frame (ORF) upstream of RNA-2 which partially overlaps the large ORF and encodes a protein of unknown function that displays a great degree of sequence diversity with other torradoviruses [Tomato marchitez virus (ToMarV), Tomato chocolàte virus (ToChV), and Tomato necrotic dwarf virus (ToNDV) are presently the only known spherical viruses that are transmitted by three whitefly species, i.e., Trialeurodes vaporariorum, Bemisia tabaci, and Trialeurodes abutilonea in a semi-persistent manner [B. tabaci and T. vaporariorum thrive in warmer temperatures with prolonged dry spells of up to four months , although T. vaporariorum is more tolerant of cold weather.ToTV along with three other torradovirus species i.e., t manner ,6. Verbet manner  further t manner . Jones et manner  indicateEl Niño. Provinces situated in the north experienced extremely high temperatures and prolonged periods of dry weather that resulted in millions of dollars in crop losses. During a national survey, an unprecedented whitefly infestation was observed in major tomato growing areas throughout South Africa. Tomato growers in the Limpopo province (the largest producer of tomatoes in South Africa) noticed severe necrosis/burnt-like symptoms on the leaves, stems, and fruit that resembled heat burn or possibly osmotic stress that usually results from an excess of nitrogen salts . Closer observation revealed that the “burnt like” symptoms were similar to those described by the tomato farmers in Murcia. Primary symptoms appeared as necrotic spots surrounded by chlorotic halos that began at the base of immature leaves, and advanced as stunted growth, vertical stem necrosis, and necrotic spots on the fruit resulting in significant yield losses in affected areas.South Africa recently experienced a severe drought which was exacerbated by the effects of Datura stramonium (jimson weed) samples that were collected from the Limpopo province (situated far north of South Africa). In addition, an arable weed species  red list) (http://redlist.sanbi.org/species.php?species=2595-13) growing among tomato crops in the northern part of the KwaZulu Natal province tested positive for ToTV infection . No other torradovirus species, including Tomato marchitez virus (ToMarV), Tomato chocolàte virus (ToChV), Tomato necrotic dwarf virus (ToNDV), and Tomato chocolate spot virus (ToChSV), were identified in South Africa. Additionally, all whitefly-infested symptomatic bell pepper plants that were assayed for the presence of torradovirus infections using RT-PCR were negative.RT-PCR assays showed that ToTV was only present on tomato crops and Abutilon grantii) growing among tomato crops in the northern KwaZulu Natal province (13.8%); adjacent/nearby tomato crops were not infected [A total of 316 tomato samples, 269 bell pepper samples, and 182 weed samples belonging to six botanical families  were tested for torradovirus infection. A high prevalence of ToTV was identified on tomato (71.6%) and D. stramonium (66.7%) in the Limpopo province of South Africa [Globally, ToTV has been identified in Spain, Hungary, Poland, Canary Islands, France, Panama, Italy, Australia, Colombia, and Morocco ,16,17,18Total RNA was extracted using a Quick-RNA™ MiniPrep kit  according to the manufacturers’ guidelines. Six hundred microliters of lysis buffer was added to 20 mg of frozen leaf tissue in a sterile 1.5 mL microcentrifuge tube containing five to six tanzanite beads. Samples were subsequently macerated using a bead beater homogenizer. Prior to the final elution step, 30 µL of nuclease-free water was added to the column and allowed to incubate for 2 min at room temperature. The quality and quantity of each RNA extract was measured using a Nanodrop 2000 spectrophotometer . Samples were stored at −80 °C pending further analysis.A two-step RT-PCR approach was used as the first measure to detect torradovirus infections due to the lack of commercially available antibodies. Template RNA (4 µL) was incubated at 65 °C for 5 min and subsequently kept on ice. A master mix component containing 2 µL of a gene-specific primer, 4 µL of reaction buffer, 1 µL of reverse transcriptase enzyme, 1 µL of ribolock RNase inhibitor, 2 µL of dNTPs, and 6 µL of nuclease-free water was added to make a final volume of 20 µL for each reaction. cDNA was synthesized using a RevertAid Premium Reverse Transcriptase kit  according to the manufacturers’ guidelines. Conditions for RT were 42 °C for 1 h and 70 °C for 10 min.PCR was carried out in 20 µL reaction volumes using a KAPA2G Fast HotStart ReadyMix PCR kit . Each PCR reaction contained 10 µL of KAPA Ready Mix, 2 µL of each primer (10 ng/µL), 30 ng of template DNA, and nuclease-free water. A set of degenerate torradovirus primers Torrado-2F ; nt 2528–2547 of ToMarV ; nt 2561–2580 of ToChSV (GQ305132) and nt 2568–2587 of ToChV ) and Torrado-2R ; nt 3023–3042 of ToMarV ; nt 3056–3075 of ToChSV (GQ305132) and nt 3063–3082 of ToChV ) that target a 515 bp region (overlapping the Vp35 and Vp26) located on the RNA-2 strand was used to detect the presence of torradoviruses [Escherichia coli cells were transformed by heat shock (42 °C for 30 s) prior to a 30 min incubation on ice. Successful transformants were selected using blue/white colony screening and cultured overnight at 37 °C in Luria–Bertani (LB) broth containing 50 µg/mL kanamycin. Plasmid extractions were carried out using a Zyppy Plasmid Miniprep Kit . The insert was confirmed using EcoR1 endonuclease activity . Reactions were incubated at 37 °C for 15 min and terminated at 85 °C for 5 min. Bi-directional sequencing of positive transformants were carried out at Inqaba Biotec  using a 3500xL Genetic Analyzer . The Blast tool in MEGA X software [PCR-positive amplicons were excised and purified using a Zymoclean Gel DNA Recovery Kit . A TA cloning kit  was used to ligate the target sequences from purified gel extracts onto a PCR 2.1 cloning vector following the manufacturers’ guidelines. Chemically competent software  was usedSolanum lycopersicum Mill.) plant collected from the Limpopo province (DMS: 23°38′22.6104″ S 30°4′40.2996″ E) and analyzed using high-throughput sequencing (HTS). HTS data were generated using an Illumina HiSeq 2500 Ultra-High-Throughput Sequencing System  at the Agricultural Research Council Biotechnology Platform ), and the raw data were deposited into GenBank SRA: SUB8159355. Read lengths less than 25 nucleotides were trimmed and pair-end sequence libraries were generated. The raw data were trimmed using Trimmomatics version 0.36 whereby the low-quality sequence regions and Illumina universal adapter sequences were trimmed and removed. CLC genomics workbench 9.5.3 (https://www.qiagenbioinformatics.com/) was used to remove host data by matching sequence reads to a reference tomato genome  prior to de novo assembly. The remaining contigs were then identified using Blast version 2.6.0. against the NCBI nucleotide database. Contig sequences with viral identities were extracted for functional annotation with Blast2GO using the default parameters. Protein translation and ORFs were identified using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). A comparison of nucleotide and amino acid similarities was established using SIAS tools (http://imed.med.ucm.es/Tools/sias.html), and phylogenetic analysis of the RNA-1 and RNA-2 genome of ToTV was inferred from trees generated using a best-fit model in MEGA 6 software. The sequence of each RNA segment generated from the HTS analysis was reconstructed and verified with primers designed using SnapGene v5.1.4.1  from a ToTV-positive tomato (v5.1.4.1 . The genTomato spotted wilt virus (TSWV) by local tomato growers and government extension workers. Nearby weeds infested with whiteflies exhibited symptoms such as stunting and leaf deformation. . Based on phenotypic analysis, both D. stramonium in the Limpopo province of South Africa based on their nucleotide sequence similarity. Blast analysis showed that the isolate of ToTV from South Africa  matched 99% to the Polish isolate Walʹ03 (EU563947) [Abutilon grantii  growing among tomatoes in the northern KwaZulu Natal province  was not identified on tomato crops and matched 92.8% to the isolate T795 (KX132809) from Italy [Each 515 bp PCR positive product was validated by cloning and sequencing. A consensus sequence was derived from the isolates of ToTV infecting tomato crops and U563947) ,21. The The full genome of ToTV was elucidated using high-throughput sequencing. The dataset contained 23,624,259 raw paired-end reads. A total of 127,654 contigs were generated from the de novo assembly. Only 2101 contigs did not align with the host genome and consisted of viral, bacterial, fungal, and traces of plant sequences. Of the 2101 contigs, 68 contigs were similar to known viral sequences. The longest contig obtained for the RNA-1 and RNA-2 genome of ToTV was comprised of 7420 and 5381 nucleotides (nt), respectively. Sequences generated from each RT-PCR clone  was 7420 nt in length excluding the poly (A) tail and comprised of a 109 nt 5′ untranslated region, a single open reading frame (ORF-1), and an 834 nt 3′ non-coding region . ORF-1 . Their fRNA-2  spanned a total of 5381 nucleotides (nt) excluding the polyadenylated tail and comprised of two ORFs, a 172 nt 5′ leader sequence and a large non-coding region (1092 nt) at the 3′ end . ORF-1  show thaPhylogenetic analysis of the full-length RNA-1 and RNA-2 nucleotide sequences showed five distinct clades representing the five known members of the torradovirus genus that are capable of infecting tomatoes ; the taxThe full-length RNA-2 genome of ToTV from South Africa (ToT-186) did not cluster with other ToTV isolates. The tree topology in Similar tree topologies of the full-length RNA-1 and RNA-2 genomes of tomato-infecting torradoviruses are illustrated in Phylogenetic analysis of the torradovirus RNA-1 polyprotein showed that the isolate of ToTV (ToT-186) from South Africa clustered with ToTV Wal03 from Poland; this relationship is supported by a weak bootstrap value . InteresThe tree topology from the phylogenetic analysis of the torradovirus RNA-2 polyprotein shows that ToTV isolates form a distinct clade from other tomato-infecting torradoviruses . The isoThe recent ToTV outbreak in the northern Limpopo province of South Africa caused substantial damage to fields of commercially grown tomatoes . In the Potato virus Y (PVY), PepMV and ToCV were initially identified on samples that were analyzed using high-throughput sequencing. Some tomato crops exhibited burnt-like/necrotic spot symptoms typically associated with torradovirus disease, whilst others displayed interveinal leaf chlorosis and chlorotic flecking symptoms typically associated with crinivirus infections. Symptom expression may be linked to the primary infecting virus and factors that influence viral fitness. Although evidence suggests that there are no significant associations among these viruses (ToTV + PepMV and ToTV + ToCV) [In South Africa, tomato crops and weeds that tested positive for ToTV infections were often coinfected with PepMV and ToCV. These co-infecting viruses, i.e.,  + ToCV) , the symPrior to this study, whitefly-transmitted torradovirus disease was unfamiliar to South African farmers and agricultural extension workers. These symptoms were often mistaken for physiological disorders that generally result from the prolonged exposure of crops to higher temperatures or excessive pesticide applications. For many South African farmers, torradovirus infections appeared as an unrelated physiological condition and therefore requires the use of molecular assays to validate symptomatology in the field.The symptoms associated with ToTV infections are almost indistinguishable from those of other tomato-infecting torradovirus species. Therefore, a generic set of primers  was usedD. stramonium and tomato crops (Abutilon grantii) growing among tomato field crops in the northern KwaZulu Natal region was not detected on the nearby tomato crops  from South Africa matched 99% with Spanish (DQ388879) and Polish (Kra (KJ940975)) isolates. Phylogenetic analysis of the full-length RNA-1 genome showed that ToT-186 grouped with other ToTV isolates but was also the most evolutionary diverse . RNA-2 . The evo from SouComparisons of the torradovirus RNA-1  and RNA-The representation of the phylogenetic trees in Bemisia tabaci and two Trialeurodes species. Their coexistence in some locations may be an adaptive or acquired trait or possibly a type of species coevolution that is not entirely suppressing or limiting to the other. It also indicates a level of heightened fitness or the ability to adapt and thrive in the face of competition, extreme weather events, and extensive pesticide use. Weeds need to be managed more effectively, considering their role as alternative hosts. Resistant tomato varieties provide effective control against ToTV infections. These genes can also be introgressed into commercially desirable germplasm via breeding programs. In addition, good crop husbandry, intensive scouting, trap crops, fine mesh nets, mineral oils, pheromones, sticky traps, and pesticides can be used in varying combinations to manage the spread of ToTV. The use of pest phenology models may serve as an early warning system for farmers, especially those who cultivate field-grown vegetables. Importantly, there needs to be a consensus among researchers, government extension workers, farmers, and policymakers so that effective strategies can be implemented based on detailed research outputs and stringent policies that regulate the movement of seed and plant material into South Africa.Efficient control methods can be designed to contain the spread of ToTV into other provinces of South Africa. Vector control is a key management strategy for emerging viruses such as ToTV which are naturally transmitted by In this study, the prevalence and distribution of ToTV in South Africa are reported. In addition, a comprehensive analysis of the genome of a ToTV isolate from South Africa was generated using high-throughput sequencing technology. The development and use of such technology have led to scientific breakthroughs in many areas of research and may facilitate the identification of many new and emerging diseases. Such was the case of non-tomato-infecting (NTI) torradoviruses. Identification and characterization are the first steps toward developing methods to contain and possibly mitigate diseases such as ToTV. In countries such as Poland and Hungary, ToTV has been totally eradicated.


The spatial distribution of extreme rainfall cannot be seen if only using a rain-gauge observation in this location. The spatial distribution of extreme rainfall is needed to identify the impact of rainfall on landslides in large areas. The study aims to (1) develop the reconstruction of the spatial distribution of extreme rainfall using weather radar and (2) investigate the trigger that caused extreme rainfall by analyzing the synoptic-scale tropical waves. Each weather radar datum is saved in a Constant Altitude Plan Position Indicator (CAPPI). To get rainfall information, the CAPPI must be derived from Quantitative Precipitation Estimation (QPE) values. In this paper, we derived CAPPI using a Marshall-Palmer reflectivity-rain rate relationship. The result shows that rainfall formed on September 20, 2017, 21.00 UTC with total daily rainfall ranged between 176 and 247 mm in both districts and the mean of total daily rainfall has exceeded the average of monthly rainfall. The analysis of tropical waves suggests that only Kelvin waves were active and served as a possible trigger factor while the Madden-Julian Oscillation (MJO) and Equatorial Rossby (ER) waves were inactive during this extreme rainfall.Extreme rainfall accompanied by strong winds hit the province of Bengkulu in the western coastal area of Sumatera Island during September 19-20, 2017, causing floods and landslides in Seluma and Central Bengkulu district. This extreme rainfall was recorded by Bengkulu Meteorological Station about 257.0 mm day On 20 September 2017, an extreme weather event occurred in the province of Bengkulu on the western coast of Sumatra Island causing floods and landslides in several areas such as Kampung Bahari, Bengkulu, Padang Pelasan and Ngalam villages in Seluma district, and Tanjung Raman in Central Bengkulu district. Extreme weather is very likely to occur in the region of Bengkulu Province due to topographic contours and geographical location. The growth of convective clouds is one of the causes of extreme weather in the region which is related to regional and local factors such as tropical cyclone effects, eddy, shear line regions, and coastal shape [During the extreme event, observation in the Bengkulu Meteorological Station recorded the maximum temperature of 26°C while the range of maximum temperature in September is about 29°–32°C. For rainfall measurement, it reached 257.0 mm per day at the Meteorological Station Fatmawati Bengkulu while at Pulau Baai Climatological Station it reached 230.2 mm per day. These values are higher than the monthly average rainfall of September which is only about 220 mm/month for climatology of 1981–2010 so that these are classified as extreme rainfall . The limR) by utilizing reflectivity data (Z). Weather radar can be used to provide early warning and analysis of extreme weather phenomena such as heavy rain events, tornadoes, gusty winds, and wind shear [C-band weather radar is capable of detecting short-term weather conditions  and has a high resolution and wide coverage area up to a radius of 240 km over the Bengkulu area. Weather radar measures electromagnetic radiation from rain clouds and has the potential to estimate rainfall intensity   with Gematronik type and maximum radius coverage of 240 km . Data ar0 log Z) . For comA python-based open-source software Wradlib was utilized to process weather radar data. Wradlib has been widely used in the processing of weather radar data and its application , 7. It hReading the data format, defining the coordinates on the map: Wradlib can be used to process weather radar data of volumetric formats from Rainbow Gematronik. The volumetric data from the radar was then processed and saved into a NetCDF format in Cartesian coordinates. This python programming library was developed by Postdam University and University of Stuttgart.Removal of clutter caused by nonmeteorological factors such as the presence of objects on the surface of the earth  or objects in the air  by clutter filters developed by Gabella and Notarpietro . After tGridding: The reflectivity data are displayed on each vertical slope angle of the radar automatically to calculate the value of Constant Altitude PPI (CAPPI), i.e., the horizontal radar reflectivity display at a certain fixed height, and also the maximum CAPPI (CAPPIMAX) value in altitude column. The CAPPI calculation specification has been designed to have a horizontal resolution of 0.5 km/pixel and a vertical resolution of 0.5 km from 0.5 to 10 km height column.Z) into rainfall intensity (mm/hr): estimated rainfall or QPE was derived from a common Z-R relationship of Marshall-Palmer with Z = 200R1.6 for general precipitation [Conversion of reflectivity , Equatorial Rossby (ER) wave, and Kelvin waves.Cumulonimbus clouds that caused extreme weather in the Bengkulu area were detected developing over the ocean. Rain clouds were moving from the west (Indian Ocean) to the east (Sumatra Islan) as shown in Figures th September 2017 at 21 : 06 UTC (20th September 2017 at 04.06 LT) is estimated to be 60–65 dBZ. This range of reflectivity values indicates the possibility of precipitation in the form of heavy rainfall. The greater the reflectivity value, the greater the intensity of the rainfall. th September 2017 at 03 : 06 UTC (20th September 2017 at 10 : 06 LT), MCS is already in the Bengkulu region and covers the central area of Bengkulu to the north. The visible reflectivity value is not as large as the MCS in the water area, but the distributed reflectivity value distribution is between 35 and 55 dBZ in the land area. There is an interval of about 6 hours for MCS coming from moving waters covering the area of Bengkulu Province. The closure of long-term MCS in the region resulted in continuous rainfall that caused flooding in areas such as Kampung Bahari Bengkulu, Padang Pelasan and Ngalam Village in Seluma District, and Tanjung Raman in Central Bengkulu Regency. Reconstruction of total rainfall that hit the Bengkulu region was done by converting the reflectivity (dBZ) of CAPPIMAX product into rainfall intensity (mm/hr). Estimated rainfall rates are derived from two general Marshall-Palmer (MP) Z-R relationships with Z = 200R1.6 for general precipitation. The accumulated rainfall is calculated for one day from 19 September 2017 at 12 : 00 UTC to 20 September 2017 at 12 : 00 UTC. The results show the entire area of Bengkulu rain with varying intensity, where the highest total rainfall occurred in the waters of the Indian Ocean west of Bengkulu to reach 390 mm/day which is 20–30 km from Bengkulu.In Accumulated rainfall simulation results show that the phenomenon of MCS that caused the occurrence of extreme rain accompanied by strong winds hit the entire west coast of Bengkulu and concentrated in the central Bengkulu region, while for the highest rainfall intensity it is in the waters not far from downtown Bengkulu. QPE simulation also produces rainfall that can describe the situation on the occurrence of floods and landslides in the area of Bengkulu, where there is no weather observation near the area.At the scene of a flood and landslide in Bengkulu region, simulation of rainfall accumulation resulted from the conversion of radar reflectivity of CAPPIMAX product into QPE value. Simulation of temporal QPE value at the location of the flood shows the rain began to fall at 21 : 00 UTC on September 19, 2017, with a daily accumulation of 236 mm per day in Kampung Bahari Kota Bengkulu, 247 mm per day in Padang Pelasan, 217 mm per day at Ngalam Village Seluma District, and 176 mm per day in Tanjung Raman, Central Bengkulu Regency.From the graph in On 19-20 September 2017, heavy rainfall triggered floods and landslides on the Indonesian island of Sumatra, particularly in Bengkulu Province . The synoptic-scale climate variability was analyzed to determine the possible trigger factor, particularly the intraseasonal tropical waves such as Madden-Julian Oscillation (MJO), Equatorial Rossby Wave (ER), and Convectively Coupled Kelvin Wave (CCKW). During this period, the MJO was not active over the eastern Indian Ocean and Sumatra Island  moving eastward faster than MJO and passed through the Bengkulu and the south Sumatra Island on 19-20 September 2017  ZR relations calculated for one day from 19 September 2017 at 12 : 00 UTC to 20 September 2017 at 12 : 00 UTC shows the entire area of Bengkulu rain with varying intensity, where the highest total rainfall occurred in the waters of the western Indian Ocean Bengkulu to reach 390 mm per day which is 20–30 km from Bengkulu. Temporary QPE values using weather radar have been able to reconstruct the occurrence of extreme rainfall in the area of flood and landslide locations with daily rainfall accumulation of 236 mm per day in Kampung Bahari, 247 mm per day in Padang Pelasan, 217 mm per day in the Ngalam Village, and 176 mm per day in Tanjung Raman. The analysis of tropical waves suggests that only Kelvin waves were active and serve as a possible trigger factor, while the MJO and ER waves were inactive during the event over Bengkulu.Limitations of surface rain intensity measuring devices led to measurement and analysis of extreme precipitation events causing floods and landslides in Bengkulu using QPE from C-Band Doppler weather radar. The results of atmospheric dynamic reconstruction on September 19-20, 2017, using CMAX radar products show that the MCS moving from the ocean to the mainland with the instability reaches 65 dB


Lys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNA Lys3 forms 18 base pairs with complementary residues in the primer binding site (PBS)  are necessary for efficient reverse transcription and tRNALys3 selection ,3Lys3 foelection ,5,6,7,8.on [Lys3 ,10,11. Ton [Lys3 ,13. A sion [Lys3 ,14.Lys3 in the annealed complex. However, based on different types of experimental systems and different viral strains, there are conflicting models about the exact nature of these extended interactions. In vitro and in situ probing of the annealed RNA complex of HIV-1 subtype A revealed that the A-rich loop residues of the vRNA formed base pairs with residues in the anticodon and anticodon stem of tRNALys3 [Lys3 [Lys3 complex remain unclear.Numerous efforts have been spent to identify the extended interactions between vRNA and tRNAtRNALys3 ,15, and tRNALys3 ,8. HowevtRNALys3 ,17,18. ItRNALys3 ,19,20,21s3 [Lys3 ,23,24. Ts3 [Lys3 ,24,25. Hs3 [Lys3 . Additios3 [Lys3 , suggestLys3 anticodon interaction. Consistent with previous studies [Lys3 anticodon interaction in HIV-1 subtype A RNA resulted in an increased affinity for RT and enhanced the reverse transcription efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), the RNaseH activity of RT degraded the annealed vRNA: tRNALys3 complex, and the complex with a disrupted A-rich loop: tRNALys3 anticodon interaction underwent an accelerated degradation. Consistently, the loss of vRNA integrity was observed in virions carrying A-rich loop mutations. We also reported similar results in HIV-1 subtype B RNA, that the A-rich loop: anticodon interaction affected the RT-binding affinity, primer extension efficiency, and RNA integrity. Additionally, we show that the nucleocapsid (NC) protein was required to promote the extended vRNA: tRNALys3 interaction under in vitro conditions. Together, our results suggest a conserved mechanism among various HIV-1 strains: that the extended vRNA: tRNALys3 interaction protects the vRNA in the mature virions prior to entering a new host cell.Here, we present NMR evidence supporting the A-rich loop: tRNA studies , abolishLys3 . Several characteristic peaks belonging to the stem of the A-rich loop hairpin disappeared or shifted upon tRNA annealing. In particular, A155.H2 and A174.H2 gave rise to characteristic up-field shifted chemical shifts in the PBS segment RNA, and they disappeared upon tRNALys3 annealing  was synthesized by in vitro T7 transcription to investigate the possible extended interactions between vRNA and tRNALys3 a. The NMLys3 . To testtA, Lys3 c by incutA, Lys3 g, and thl change . Spectrannealing h. The NMnnealing b. The munchanged h. NMR stTo test whether the mutations of the A-rich loop affect the virus infectivity, a chimeric molecular clone containing the HIV-1 MAL 5′ untranslated region (UTR) and NL4.3 backbone  under a cytomegalovirus (CMV) promoter ) was madLys3 interaction is likely to affect RT loading and reverse transcription initiation [Since the disruption of the extended vRNA: tRNAitiation ,8, the ritiation . ConsistLys3 complex, we employed microscale thermophoresis (MST) to measure the RT-binding affinity. Since reverse transcription initiates on the genomic RNA after packaging, a 5′UTR construct (nt 1–358) favoring the dimer conformation with a packaging signal exposed was prepared to represent the vRNA [Lys3 at 50 nM and a 1:1 ratio. RT was titrated to reach a final concentration ranging from 0.00252 µM to 20.6 µM in the WT complex samples and 0.000763 µM to 6.2 µM in the mutant complex samples. The fluorescence signal changes upon RT titrations versus the RT concentrations that were plotted  +179 nt (cDNA product)). Primer extension products were resolved by polyacrylamide gel electrophoresis and imaged by fluorescent scanning cDNA in infected cells , we hyposcanning c. It appscanning d. These Lys3 complex with A-rich loop mutations was favored by RT and exhibited enhanced efficiency in producing cDNA products. Early reverse transcription may take place in mature virions, but the rate is limited by the dNTP concentrations in the extracellular environment [Lys3 to visualize the degraded RNA fragments from the 3′-end. The appearance of a degradation band slightly higher than 156 nt was observed in both the WT and mutant but was absent when no RT was added  before or during reverse transcription. However, the in vitro binding and primer extension results show that the vRNA: tRNAironment ,32,33,34ironment . Therefoas added b, indicaactivity ,37,38  to generate PBSm-NL4.3, in which the unpaired adenosines were substituted by UA pairs a,b. The Lys3 in vitro and investigated the complex structure by EMSA and NMR. One of the unexpected findings was that the structure of the annealed complex was different, depending on the annealing conditions. NC-promoted annealing was performed by mixing the PBS-NL4.3 RNA and tRNALys3 at a 1:1 ratio with various amounts of NC at 37°C. NC proteins were removed from the RNA complex by high salt washes , and the annealed complexes were analyzed by EMSA. Under all conditions, a stable vRNA: tRNALys3 complex was formed in the absence of Mg2+  overnight showed a weak slow-migrating band (slow-complex 1) , PBSm-NL4.3: tRNAmide gel e. Both ttRNALys3 e. These  complex f, demonsnnealing b.Lys3 complex structure, we noticed that both the in vitro annealing conditions led to the same complex structure in the MAL PBS segment and 5′UTR  = 46.5 ± 29 nM, Lys3 complex  substituting GUAAAA nt 162–167) by CUAUG and used the RNA as a template for tRNA by CUAUGon phase . Within on phase . Our daton phase . Thus, tLys3 onto the PBS segment does not occur spontaneously at a physiological temperature, as both the PBS segment and tRNALys3 are highly structured. Complete annealing requires mature NC, which is produced after Gag is processed by viral protease, as in virio SHAPE activities of the viral RNA from protease-deficient virions did not show a similar pattern of complete tRNALys3 annealing as that of the WT virions [pro primer to expose their complementary sequences for annealing [Lys3 complex, the extended A-rich loop: tRNA anticodon interaction is formed in both heat- and NC-annealing conditions. This is consistent with the previously reported results that examined the structure of the MAL PBS segment: vRNA complexes annealed by heat and by NC using enzymatic probing [Lys3 onto the PBS-NL4.3, but both the EMSA and NMR data indicate that the structure of the complex was not the same as the NC-annealed complex  of approximately 90 nM for RNA/RNA and 5 nM for DNA/RNA and DNA/DNA [d in the mutant vRNA: tRNALys3 complexes carrying A-rich loop mutations , 10 mM spermidine, and 0.01% (v/v) Triton X-100) using dNTPs (12 mM each), Ribolock RNase Inhibitor , and T7 RNA polymerase (1 µM). Transcriptions reactions were held at 37 °C for 4 h and quenched with 1-M urea and 25 mM ethylenediaminetetraacetic acid (EDTA). RNAs were purified by sequencing gels, visualized by UV shadowing, electroeluted from the gel, and washed in ultra-centrifugal filters to remove acrylamide and salts.RNA used for in vitro experiments were synthesized by T7 in vitro transcription. The DNA templates for RNA synthesis were produced by amplifying the corresponding sequences with a T7 promoter sequence. To determine optimal conditions for large-scale transcriptions, small-scale trial transcriptions were performed using varying concentrations of MgClE. coli BL21 (DE3)-pLysS cells  with 1-mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37°C, respectively. The harvested cell pellets were resuspended in lysis buffer , pH 8.0), sonicated, and centrifuged. The supernatant was applied to a cobalt column  and purified according to the manufacturer’s protocols with an extra-high-salt buffer  washing step. The protein was then subjected to size exclusion chromatography for further purification and buffer exchange and stored in buffer containing 20-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5), 80 mM NaCl, and 2 mM tris(2-carboxyethyl)phosphine (TCEP) at −80 °C. The HIV-1 NC protein expression plasmid was a kind gift from Dr. Michael Summers . The recombinant HIV-1 NC protein was expressed and purified exactly as previously described [Recombinant HXB2-RT and HXB2-RT-E478Q were expressed in escribed .4 and 62.5 mM of NaOAc at room temperature in the dark for 90 min, followed by incubating with an additional 0.42 mM Na2SO3 at room temperature for 15 min and then mixing with 0.58 mM FTSC dye (dissolved in dimethylformamide) at 37 °C in the dark for 3 h. To quench the reaction, LiCl was added to a final concentration of 0.74 M, and the sample was mixed with 2.5 volumes of 100% ethanol and incubated at −80 °C for 1 h. The sample was centrifuged at 13,000× g for 20 min at 4°C, and the supernatant was discarded. The precipitated RNA was washed twice with 75% ethanol and lyophilized for 10 min. The lyophilized RNA was dissolved in double-distilled H2O and stored for further use.Cy3-labeled tRNA was prepared as described previously . 3′-FTSC1H NMR experiments was prepared in 50–200 µM and dissolved in a 2H2O buffer containing 10 mM deuterated Tris-HCl (pH 8.0) and 2 mM MgCl2. RNA used for 2D 1H-1H Nuclear Overhauser Effect Spectroscopy (NOESY) experiments were prepared at 200–300 µM in the same buffer. The NMR data were collected at 308 K on a Bruker Avance III 800 MHz spectrometer equipped with a TCI cryoprobe. Data was processed with NMRPipe and NMRDraw using the NMRBox cloud-based virtual machine [RNA used for 1D  machine . Data waLys3 at a 1:1 ratio in the annealing buffer  and incubated at 94 °C for 5 min, 85 °C for 15 min, 75 °C for 15 min, and 65 °C for 60 min. NC annealing was carried out by mixing HIV-1 vRNA with tRNALys3 at a 1:1 ratio and mixing with various amounts of NC, as indicated in the Results section. NC was then removed from the RNA complex by high-salt washes with 10 mM Tris-HCl, pH 7.5, 1.5 M NaCl, and 1 mM MgCl2) for the in vitro analysis.Heat annealing was carried out by mixing HIV-1 vRNA with tRNALys3, and 50 nM of the complex was titrated with RT at concentrations varying from 0.00252 µM to 20.6 µM for the MAL 5′UTR:tRNALys3 experiments and 0.000763 µM to 6.2 µM for the MAL 5′UTR-MutA:tRNALys3 experiments. Experiments were performed using a Nanotemper Monolith and were analyzed using MO.Affinity Analysis . Each experiment was repeated three times.3′-FTSC-labeled WT and mutant MAL 5′UTR RNA samples were pre-annealed to tRNALys3 in annealing buffer using NC (vRNA: tRNA: NC = 1:1:12). NC was removed by high salt washes, as described in Lys3 experiments and 0.000763 µM to 25 µM for the PBSm-NL4.3: tRNALys3 experiments. Experiments were performed using a Nanotemper Monolith and were analyzed using MO.Affinity Analysis . Three independent replicates of the measurements were performed for the Kd quantification. For the biphasic binding curve, Kd was determined by excluding data points for the low-affinity binding mode during the analysis of the high-affinity binding mode and vice versa [3′-FTSC-labeled WT and mutant NL4.3 RNA were annealed to tRNAce versa .Lys3 in an annealing buffer by heat and by NC, respectively. The annealed complexes were mixed to 90 nM with 3 mM MgCl2, 1 µM NC, Ribolock RNase Inhibitor  0.6 units, 2.7 µM RT, 25 mM HEPES, and 80 mM NaCl. To initiate the reaction, dNTPs were added to 90 µM and held at 37 °C. Each time point was quenched with 20 mM EDTA and placed on ice. SDS was added to 12.5 mg/mL, and samples were boiled for 5 min. Proteins were removed by phenol–chloroform extraction, and the RNA samples were boiled for 3 min and loaded onto an 8% denaturing 8 M Urea polyacrylamide gel. The gels were visualized by fluorescent scanning on a Typhoon FLA 9000 .The MAL and NL4.3 vRNA were annealed to 3′-Cy3 tRNALys3, as described in E478Q in a buffer containing 25 mM HEPES, 80 mM NaCl, and 0.6 units of Ribolock RNase Inhibitor . The reactions were incubated at 37 °C for the specified times and quenched by the addition of EDTA. SDS was added to 12.5 mg/mL, and samples were boiled for 5 min. Proteins were removed by phenol–chloroform extraction, and the RNA samples were boiled for 3 min and loaded onto an 8% denaturing 8 M Urea polyacrylamide gel. The gels were visualized by fluorescent scanning on a Typhoon FLA 9000 .The 3′-FTSC-labeled HIV-1 vRNA was pre-annealed to tRNAThe 293FT cells were propagated in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS). WT and mutant MAL chimeric viruses were produced by transfection . Briefly5 of TZM-bl cells (AIDS Reagent Program) in 12-well plates. The infected cells were harvested 20 h post-infection and fixed with 4% paraformaldehyde. The percentage of EGFP cells were measured by Accuri Flow cytometer.The amount of pseudo-typed viruses was quantified by p24 ELISA, using precoated MicroFleur well plates . Pseudo-typed viruses containing 250 ng of Gag p24 was used to infect 2 × 105 of 293 cells in 12-well plates. The cells were harvested 2 h post-infection and subjected to DNA extraction with QIAamp DNA Blood Mini Kit . Cellular DNA (2 µL) was evaluated by qPCR using primer sets specifically targeting the reverse transcription products [Equivalent amounts (250 ng of Gag p24) of WT and mutant pseudo-typed viruses were pretreated with DNase I and DpnI and then infected 2 × 10A total of 250 µl of pseudo-typed viruses were used for RNA extraction with TRIzol Reagent  following the manufacturer’s user guide. The extracted nucleic acids were subjected to DNase I and DpnI digestion . The viral genomic RNA was extracted using a NEBNext Poly(A) mRNA Magnetic Isolation Module , and the integrity were evaluated by qRT-PCR using the primer and probe sets listed in t-test was used to compare the significance of differences between different groups with Prism . The p-values less than 0.05 were considered significant. Statistical differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. More than three biological replicates were applied in all the experiments.An unpaired


Circular RNAs (circRNAs) are a class of noncoding RNAs, generated from pre-mRNAs by circular splicing of exons and functionally largely uncharacterized. Here we report on the design, expression, and characterization of artificial circRNAs that act as protein sponges, specifically binding and functionally inactivating hnRNP (heterogeneous nuclear ribonucleoprotein) L. HnRNP L regulates alternative splicing, depending on short CA-rich RNA elements. We demonstrate that designer hnRNP L-sponge circRNAs with CA-repeat or CA-rich sequence clusters can efficiently and specifically modulate splicing-regulatory networks in mammalian cells, including alternative splicing patterns and the cellular distribution of a splicing factor. This new strategy can in principle be applied to any RNA-binding protein, opening up new therapeutic strategies in molecular medicine. Circular RNAs (circRNAs) exist in all eukaryotes investigated so far and have been known more than four decades, starting with the plant-pathogenic viroid RNAs , followeFunctionally, however, circRNAs remain largely unexplored until today, except for a miRNA sponge function, experimentally validated only for few cases ,17. As sin vitro by T7 transcription and RNA ligation, alternatively by an in vitro ribozyme-mediated processing pathway , can function either as splice activator or repressor, requiring binding to short CA-repeat or CA-rich RNA elements in its target pre-mRNAs . Here, win vitro circularization 100 is 365 nts in length; the negative control, PIE-control, contains vector sequences, producing a circRNA of 484 nts.For PIE-(permuted-intron–exon) constructs, the PIE expression cassette, which relies on group I splicing-mediated rization , was syn20, (CA)100, control, CA-SELEX X2 and CA-SELEX X4 sequences were synthesized, or PCR-amplified from the corresponding PIE constructs, and cloned between the NotI and SacII sites of pAV-U6+27-Tornado-Broccoli, replacing the Broccoli aptamer sequence. The SELEX X2 and X4 constructs contain two or four copies, respectively, of a 20-nts CA-rich sequence, derived from our initial SELEX study and validated as a high-affinity target sequence of hnRNP L , Tornado CA-SELEX X2 (87 nts), and Tornado-CA-SELEX X4 (149 nts), were overexpressed. As an additional control, the vector pAV-U6+27-Tornado-Broccoli, expressing a circRNA of 96 nts, was used. The Tornado-(CA)100, the Tornado-control, and the Tornado-CA-SELEX X4 constructs were also made with the Broccoli aptamer, by cloning into the KflI site of the pAV-U6+27-Tornado-F30-Broccoli vector, resulting in circRNAs Tornado-(CA)100 , the Tornado-control , and the Tornado-CA-SELEX X4 .For Tornado circRNA expression, pAV-U6+27-Tornado-Broccoli and pAV-U6+27-Tornado-F30-Broccoli vectors were used Addgene; . Inserts100 unit [see above under PIE-(CA)100], or the negative control sequence, were cloned between the EcoRV and SacII sites of the pcDNA3.1 (+) ZKSCAN1 MCS exon vector  and flanking inverted repeats  repeats  into the repeats , resultiFor a complete list of DNA- and RNA-oligonucleotides, see in vitro transcription, using double-stranded DNA-oligonucleotide templates and the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB), and labeling internally by incorporation of azide-modified UTP analog , followed by RQ1 DNase treatment  and Sephadex column purification (Sigma-Aldrich). For circularization, T4 RNA ligase  was used, followed by biotin labeling with copper-free Click Chemistry .RNAs were synthesized by in vitro binding assays, 50 pmol of biotinylated RNA was prebound to 30 μl MyOne Streptavidin C1 Dynabeads , followed by incubation with 20 μl HeLa cell lysate (corresponding to 2.2 × 105 cells) and washing off unbound proteins at 300 mM KCl. Bound proteins were released and separated by 10% SDS-PAGE, followed by Western blotting with hnRNP L, GAPDH (Sigma-Aldrich), or IMP3 (Millipore) primary antibodies and peroxidase-coupled secondary antibodies (Sigma-Aldrich).HeLa cells were lysed in RIPA buffer . For in vivo, antibodies were added to pre-cleared lysate , incubated overnight at 4°C, followed by addition of 30 μl (1 mg) Protein A or G Dynabeads (Thermo Fisher Scientific), and rotation for two hours at 4°C. Protein–RNA complexes were washed by increasing the stringency up to 600 mM NaCl. RNA from input and immunoprecipitated fractions was extracted by TRIzol (Ambion), followed by reverse transcription  and (q)PCR with gene- and linear/circular-specific primers. The fraction of bound target RNAs was calculated for each target relative to the corresponding input fraction.For assaying hnRNP L sponging in vitro , followed by RQ1 DNase treatment (Promega) and purification by the Monarch RNA Cleanup Kit (New England Biolabs). Ribozyme-catalyzed circularization was induced in splicing buffer  by the addition of GTP , followed by incubation for 8 min at 55°C. Circularization efficiency was checked on the 2% E-Gel system (Thermo Fisher Scientific). To purify circRNAs further, RNA was treated with RNase R (Lucigen) and HPLC-fractionated -mediated circularization in vitro , RNA wasRNAs were analyzed directly by electrophoresis in denaturing polyacrylamide gels , agarose gel (1.5%), or by E-gel electrophoresis , and stained by SYBR Gold; in case of Broccoli-carrying RNAs, gels were first stained with DFHBI . For RNA analysis, either low- or high-range RNA markers were used (Thermo Fisher Scientific).4 HeLa cells were seeded in 24-well plates one day before transfection, and 100 or 500 ng linear or circular RNA were transfected, using Lipofectamine™ MessengerMax™ mRNA transfection reagent (Thermo Fisher Scientific), and harvested after 24 h. RNA was isolated using TRIzol (Ambion) and RNeasy columns (Qiagen), followed by reverse transcription  and alternative splicing assays by PCR with gene-specific primers.For transfection of PIE circRNA, 5 × 106 cells per plate) one day before transfection. Transfection was performed by the TurboFect reagent (Thermo Fisher Scientific). After 48–72 h, total RNA was isolated using Norgen kit (Norgen Biotek). For the time course experiment, RNA was isolated 1, 2, 3 and 4 days post-transfection. For alternative splicing assays, total RNA (1 μg) was primed by oligo (dT)20 and reverse-transcribed , followed by PCR assays using gene-specific primers , followed by Western blot analysis for hnRNP L (Sigma-Aldrich), GAPDH (Sigma-Aldrich), and hnRNP A1 (Santa Cruz Biotechnology). Proteins were quantified by densitometry, using the ImageJ software, and based on biological replicates. In addition, the nucleo-cytoplasmic distribution of circRNAs and linear RNAs was determined by RT-qPCR.100, CA-SELEX X4, and CA-SELEX X4/Broccoli circRNAs. 50 ng of the RNA transcript and 200 ng total RNA as competitor was reverse-transcribed (see above). Based on that, a standard curve (five 10-fold dilutions from 5 to 0.0005 ng) was derived to determine absolute quantities using the NEBiocalculator software. Real-time PCR was carried out using Luna® Universal qPCR Master Mix (NEB) on an Eppendorf realplex2 thermocycler. Standard-curve R2 values were >0.99 and amplification efficiency between 90% and 100%.To determine the absolute concentration of circRNAs (copy number per cell), total RNA was isolated from HeLa cells, using TRIzol (Ambion) after transfection of Tornado-expression plasmid DNA, or using TRIzol (Ambion) and RNeasy columns (QIAGEN) after transfection of RNAs expressed by the PIE system. Total RNA (200 ng) was reverse-transcribed by qScript reverse transcriptase (Quanta) followed by real-time PCR carried out in triplicates. As quantitative standards, we used control, (CA)4 cells/μl) on a 10% SDS-polyacrylamide gel, and comparing signals obtained with recombinant GST-hnRNP L protein .To determine the corresponding concentration of hnRNP L, HeLa cell lysate was prepared as described above and analyzed by Western blotting for hnRNP L and GAPDH (see above), using 1, 2.5, and 5 μl 100 or control circRNA, using Lipofectamine™ MessengerMax™ mRNA transfection reagent (Thermo Fisher Scientific). For RNAi-knockdown, 8.8 × 105 cells were reverse-transfected with RNAiMAX (Thermo Fisher Scientific), using siRNAs specific for human hnRNP L and luciferase GL2 (Sigma-Aldrich). 72 h post-transfection, total RNA was isolated, quality-controlled and depleted of ribosomal RNA (NEBNext rRNA depletion kit), followed by library preparation  and sequencing on Illumina NextSeq 500 . RNA-seq data were deposited in the Sequence Read Archive (PRJNA610182) of NCBI.For global analysis of sponging, 2.5 × 10http://www.gencodegenes.org) was applied for gene expression and splicing analyses. From each of the six sponge samples, ∼81% (81.01–82.15%) of the sequenced reads (51.37–59.91 mio) were uniquely mapped, in the two siRNA-knockdown samples, ∼77% of the sequenced reads (62.53 and 74.22 mio).Sequence reads were aligned to the human genome sequence (hg19 assembly) using STAR . The comFor global gene expression analysis, the read coverage of annotated protein coding genes were normalized with the number of uniquely mapped reads in each sample and with the mRNA length. Normalized read coverage from ∼9200 genes, of which at least one sample had a minimum read coverage of 16, was used to calculate the expression ratios between samples  were calculated as following:For predicting increased single exon skipping upon CA-sponging by circRNA, junction read counts for exon skipping and inclusion were used to calculate the ratio of skipping versus inclusion events . We firs2 (sk:incl_CA100_0.5 μg) – log2 (sk:incl_control_0.5 μg);R_0.5 = log2 (sk:incl_CA100_1.0 μg) – log2 (sk:incl_control_1.0 μg);R_1.0 = log2 (sk:incl_CA100_2.5 μg) – log2 (sk:incl_control_2.5 μg).R_2.5 = log100 samples. For the prediction of increased single exon inclusion upon CA-sponging by circRNA, the analogous procedure was applied for the ratio of exon inclusion versus skipping events (incl:sk).Positive targets were selected based on the dose-dependent increase in skipping/inclusion effects after circRNA transfection (R_1.0 – R_0.5 > 0.75 or R_2.5 – R_0.5 > 0.75); in addition, to reduce the false positive ratio, a minimum skipping junction read counts of 16 was required in the (CA)R) were calculated between samples with hnRNP L-specific versus luciferase-control siRNA, and positive targets were predicted, if R values were >0.75. In addition, a minimum of 16 skipping junction read counts was required.For the siRNA-knockdown approach, corresponding ratios 15, and (CA)20. As an alternative option, a 20-nts CA-rich sequence was used, derived from our earlier SELEX study and validated as a high-affinity target sequence of hnRNP L . As a result, we were able to predict target exons that responded in their inclusion or skipping pattern to the CA-sponge circRNA A Figure : 107 exo20 (87 nts), as well as two (87 nts) or four copies (149 nts) of the SELEX-derived hnRNP L high–affinity RNA motif s Figure , with efTJP1 and BPTF/FALZ), monitoring exon inclusion between one to four days post-transfection , after overexpression of a corresponding Tornado-vector-based construct onstruct . HeLa ce20 and (CA)100] or oligomerized CA-rich high-affinity binding motifs; either of them bound hnRNP L in vitro and in vivo with high efficiency, resulting in alternative splicing modulation comparable to RNAi knockdown effects. The high overexpression of an hnRNP L sponge also explains the dramatic translocation of the hnRNP L protein, from a predominant nuclear to cytoplasmic localization, where circRNAs accumulate. The strong correlation between circRNA-mediated sponging and classical siRNA-mediated RNAi effects on alternative splicing networks underlines that sponging effectively inactivates the RNA-binding protein Therefore circRNA-mediated sponging of RNA-binding proteins should be considered as an alternative to RNAi-based knockdown. We were able to achieve similar effects on alternative splicing networks and the cellular distribution of an RNA-binding protein by either direct circRNA transfection or by overexpression. In sum, our results promise that designer circRNAs can be developed into a novel and highly specific new class of therapeutic RNAs, to be applied in cases where overexpressed  RNA-binding proteins cause human disease, such as in many tumor tissues. For example, hnRNP L is overexpressed in prostate tumors, resulting in extensive changes of alternative splicing patterns, including those encoding prostate tumor-specific genes such as the androgen receptor .RNA-seq data were deposited in the Sequence Read Archive (PRJNA610182) of NCBI.gkaa1085_Supplemental_FileClick here for additional data file.


Small interfering RNAs (siRNAs) are potential tools for gene-silencing therapy, but their instability is one of the obstacles in the development of siRNA-based drugs. To improve siRNA stability, we synthesised a double-stranded RNA-binding cationic oligodiaminogalactose 4mer  and investigated here its characteristics for siRNA stabilisation in vitro. ODAGal4 improved the resistance of various siRNAs against serum degradation. The effect of ODAGal4 on siRNA stabilisation was further amplified by introduction of modified nucleotides into the siRNA. In particular, a combination of ODAGal4 and incorporation of phosphorothioate linkages into the siRNA prominently prevented degradation by serum. The half-lives of fully phosphorothioate-modified RNA duplexes with ODAGal4 were more than 15 times longer than those of unmodified siRNAs without ODAGal4; this improvement in serum stability was superior to that observed for other chemical modifications. Serum degradation assays of RNAs with multiple chemical modifications showed that ODAGal4 preferentially improves the stability of RNAs with phosphorothioate modification among chemical modifications. Furthermore, melting temperature analysis showed that ODAGal4 greatly increases the thermal stability of phosphorothioate RNAs. Importantly, ODAGal4 did not interrupt gene-silencing activity of all the RNAs tested. Collectively, these findings demonstrate that ODAGal4 is a potent stabiliser of siRNAs, particularly nucleotides with phosphorothioate linkages, representing a promising tool in the development of gene-silencing therapies. Although siRNA-based therapeutics have enormous potential for silencing specific genes that cannot be targeted by existing drugs, the development of siRNA-based drugs has been faced several difficulties: prevention of rapid degradation by nucleases, delivery to the target tissue, entry into the target cells, avoidance of innate immune responses and reduction of off-target effects4.RNA interference (RNAi) is a gene regulatory mechanism in which the expression of specific genes is silenced by endogenous double-stranded RNAs or by synthetic short interfering RNAs (siRNAs) consisting of oligoribonucleotide duplexes of 21–23 bases7. For instance, phosphorothioate modification, which substitutes a sulphur atom for the non-bridging oxygen in the phosphodiester bond8, is a classical modification applied to antisense oligonucleotides9. Phosphorothioate modification is particularly effective for providing DNase resistance, and thus is commonly used in antisense oligodeoxynucleotides10; however, it is also applicable to siRNAs11. Moreover, phosphorothioate modification improves the cellular uptake of antisense oligonucleotides9 and siRNAs12 because of its hydrophobicity.To reduce their instability, most of the developed siRNA agents carry chemical modifications in their nucleotide sugars, nucleobases, or phosphodiester linkages to prevent RNase cleavageO-methyl modification, which substitutes a methoxy group for the hydroxy group at the 2′-position of the ribose moiety14, is an effective modification for preventing RNase digestion. Locked nucleic acid (LNA)16 and 2′-deoxy-2′-fluoro nucleotides18 are also potent in supressing the cleavage of RNAs. For instance, the recently approved siRNA-based drugs givosiran19 and patisiran20 are modified by phosphorothioate linkages, 2′-O-methyl and 2′-deoxy-2′-fluoro nucleotides, and by 2′-O-methyl nucleotides, respectively7. However, chemical modification is not a perfect strategy for siRNA protection: extensive 2′-O-methyl, LNA and phosphorothioate modifications may reduce the gene-silencing activity of siRNAs22. Moreover, RNAs modified with extensive phosphorothioate modification might have increased cytotoxic effects23.In addition to phosphodiester linkages, the ribose moieties of nucleotides are also widely targeted as sites for chemical modification. For example, 2′-25. In general, chemical modification aims to prevent the second step; therefore, an alternative strategy to preserve the siRNA duplex is to prevent dissociation of the double strand into single strands. RNA duplexes form an A-form helix structure in which the major grooves have higher negative potential than the minor grooves because anionic phosphates of the internucleotide bonds line the inside edge of the major grooves26. Thus, cationic molecules that can fit into the major grooves are expected to conserve the RNA duplex structure and thereby prevent RNA degradation. Indeed, aminoglycoside antibiotics with one or more protonated amino groups bind to RNA duplexes and stabilise them28, suggesting that cationic amino sugars have favourable properties for the stabilisation of RNA duplexes. For siRNA stabilisation, however, an excess amount of cationic carriers is required because of their weak affinity for siRNAs. As a consequence, these cationic carriers are prone to be cytotoxic due to nonspecific binding to other biomolecules including DNA, RNA and proteins30. Therefore, to stabilise siRNAs with low cytotoxicity, it is necessary to develop cationic molecules that can specifically bind to RNA duplexes with higher affinity.The degradation of a double-stranded siRNA by RNase occurs in two steps: first, dissociation of the double-stranded RNA into two single-stranded nucleotides; second, cleavage of the single strands by RNase33  is similar to the width of the major groove of A-form RNA double helix31, whereas that of B-form DNA double helix is much wider34. In addition, the saccharide backbones of these oligosaccharides can form curved structures which are favourable for binding to the RNA helix structure31. Therefore, these cationic oligosaccharides are expected to preferentially fit into the major groove of RNA helix and bind to the RNA duplex through interaction between the amino or guanidino groups of the oligosaccharides and the phosphate groups in the phosphodiester linkages of the RNA duplex -linked 2,6-diamino-2,6-dideoxy-d-galactopyranose 4mer   and β-2-microglobulin (B2M), named HP2 and B2M2, respectively , their half-lives were further increased. For instance, HP2-M3 and B2M2-M3, which were introduced 2′-O-methyl modification into alternating nucleotides in the sense strands and on all nucleotides in the antisense strands, showed a marked improvement in serum stability, with a respective 9.34- and 16.4-fold increase in half-life as compared with the unmodified siRNAs without ODAGal4  were longer than 30 h, corresponding to a 6.36–8.06-fold increase as compared with HP2 and B2M2 without ODAGal4. Furthermore, in the presence of ODAGal4, the RNAs completely modified with phosphorothioate linkages (HP2-S3 and B2M2-S3) were more tolerant to serum digestion than the RNAs with extensive 2′-O-methyl modification (HP2-M3 and B2M2-M3): the half-lives of HP2-S3 and B2M2-S3 with ODAGal4 were longer than 90 h, and the fold increases in half-life relative to HP2 and B2M2 without ODAGal4 were 16.6 and 17.5, respectively. Collectively, these results suggest that a combination of ODAGal4 and either 2′-O-methyl or phosphorothioate modification markedly increases the serum stability of RNAs, but the characteristics of stabilisation differ between 2′-O-methyl and phosphorothioate modifications.We next prepared and assessed the serum stability of RNAs with phosphorothioate modification 31 and α-(1 → 4)-linked 2,6-diamino-2,6-dideoxy-d-mannopyranose 4mer (ODAMan4)32, whose pyranosyl structures differ from that of ODAGal4; and β-(1 → 4)-linked 2,6-dideoxy-2,6-diguanidino-d-galactopyranose 3mer , which contains guanidino groups instead of amino groups33 -linked 2,6-diamino-2,6-dideoxy- → 4-link38. To examine whether ODAGal4 protects single-stranded RNAs against serum digestion, the sense and antisense single strands of HP2, B2M2 and their modified analogues  was more resistant to degradation than HP2 with individual modifications (HP2-M1 and HP2-S1), and addition of ODAGal4 greatly amplified its serum stability  showed a modest increase in stability with or without ODAGal4 relative to HP2. In contrast, RNAs with phosphorothioate modification on the antisense strand combined with 2′-deoxy-2′-fluoro or LNA modification on the sense strand (HP2-FS and HP2-LS) showed greatly improved serum stability even in the absence of ODAGal4. RNAs with 2′-O-methyl and phosphorothioate modifications (HP2-MS2 and HP2-MS3) also showed improved serum stability. These results suggest that a combination of modifications including phosphorothioate modification and other modifications is effective for biological stabilisation of RNAs. Adding ODAGal4 to the phosphorothioate RNAs further increased their half-lives: relative to HP2 without ODAGal4, HP2-MS2 and HP2-MS3 showed prominent serum stability , followed by HP2-FS and HP2-LS . These results demonstrate that ODAGal4 preferentially improves the biological stability of RNAs with phosphorothioate modification among chemical modifications, and that ODAGal4 and phosphorothioate modification combined with other modifications are strongly effective for siRNA stabilisation.It has been suggested that combinations of multiple modifications in siRNAs are more effective than single modifications for protection against cleavage in serumity Fig. A, B. Nexity Fig. C, D. RNATm) of the RNAs , in agreement with our previous study33. In the absence of ODAGal4, the Tm values for 2′-O-methyl RNAs (HP2-M1 to HP2-M3 and B2M2-M1 to B2M2-M3) were higher than those for HP2 and B2M2, suggesting that the introduction of 2′-O-methyl modification enhances the thermal stability of RNA duplex structures as consistent with a previous report41. The Tm values for the 2′-O-methyl RNAs were further increased by ODAGal4 treatment (ΔTm = 0.1–3.6 °C), indicating that ODAGal4 and this modification additively enhance the thermal stability of the RNAs. In sharp contrast, the Tm values for phosphorothioate RNAs (HP2-S1 to HP2-S3 and B2M2-S1 to B2M2-S3) without ODAGal4 were lower than those for the unmodified siRNAs in good agreement with previous studies43. However, the Tm values for the RNAs containing modified nucleotides were markedly elevated in the presence of ODAGal4 (ΔTm = 2.1–7.9 °C), indicating that, although phosphorothioate modification decreases the thermal stability of RNAs, the stability of RNAs is profoundly improved when this modification is combined with ODAGal4.To determine whether ODAGal4 increases thermal stability of RNAs with chemically modified nucleotides, we next analysed the melting temperature . The presence of ODAGal4 did not apparently alter the shape of the spectra, suggesting that ODAGal4 does not change the conformation of the RNA duplex.To examine whether the thermal stabilisation of RNAs by ODAGal4 is attributed to conformational changes in the RNA duplex, circular dichroism (CD) spectra of the RNA duplexes were recorded  that binds one ODAGal4 molecule33 and its analogue with phosphorothioate linkages (12M-S1) , which cleaves the target mRNA for gene suppression4. To assess whether ODAGal4 treatment interrupts gene-silencing activity, complexes of ODAGal4 and RNAs were introduced into cultured cells and the expression of their target genes was analysed by quantitative PCR  before transfection, the knockdown efficiencies of the siRNAs were found to be more or less comparable to those without ODAGal4  additionally increase siRNA stability independent of ODAGal4 binding.We also demonstrated that RNA duplexes partially substituted with phosphorothioate linkages in the 3′-terminus improve their stability in serum, and addition of ODAGal4 further amplifies the stability. At present, it is unclear why the position of phosphorothioate modification in the RNAs is associated with biological stability. The two RNAs that were partially modified with phosphorothioate linkages in either the 5′- or 3′-terminal region had similar 52. If ODAGal4 binding to the siRNA is excessively tight, the siRNA will be unable to interact with the RISC, and consequently will not separate into single strands. We previously reported that ODAGal4 binds to RNA duplexes with higher affinity as compared with neomycin B33, one of the strongest known RNA binders among aminoglycosides53, raising the concern that ODAGal4 might interrupt the gene-silencing activity of siRNAs. Notably, however, the interaction of ODAGal4 and siRNAs did not inhibit the gene-silencing activity of any siRNAs tested, including the RNAs with chemically modified nucleotides, revealing that ODAGal4 binding to RNA duplexes is not too strong; accordingly, the siRNAs can be loaded onto the RISC. Taken together, these observations demonstrate that the strength of binding between ODAGal4 and siRNAs is in an appropriate physiological range: it is tight enough to stabilise the RNA duplex, and simultaneously weak enough to allow RISC formation followed by gene suppression. Our present findings indicate that hyper-modification with phosphorothioate linkages into RNAs have sufficient gene-silencing activity without causing cytotoxicity. These findings contrast with previous reports that excessive phosphorothioate modification may reduce gene-silencing activity21 and induce cytotoxicity23; thus, the adverse effects of this modification should be explored further. It is therefore possible that such effects depend on the conditions of gene suppression, including cell type, siRNA sequence, and the target genes. Irrespective, the combination of ODAGal4 and phosphorothioate linkages strongly improved siRNA stability in serum, even when the nucleotides were modified only in the 3′-terminal region. We thus propose that RNAs modestly modified with phosphorothioate linkages coupled with ODAGal4 may exhibit sufficient stability and gene-silencing activity, which is prone to be interrupted by severe siRNA modification22.In RNAi, siRNAs taken up by target cells are loaded onto the RISC, and then dissociate into guide and passenger strands for gene suppression54. In general, higher N/P ratios of these polycation complexes enable more effective stabilisation of the nucleotides caused by stronger interaction between the carriers and the nucleotides54. However, cell viability is gradually decreased with the increase of N/P ratios57, due to nonspecific binding of the polycations to other biomolecules. In addition, polycation complexes with excessively high N/P ratios show low siRNA efficiency for gene-silencing by interrupting siRNA release from the complexes58. These studies implicate difficulties in determining balanced N/P ratios of the polycation complexes for siRNA stabilisation with sufficient siRNA activity and low cytotoxicity. In the literature, N/P ratios of 2–100 are typically required for effective stabilisation of siRNAs by polycation complexes63. In sharp contrast, we have shown that ODAGal4 markedly stabilises siRNAs at a lower N/P ratio of 0.8 without reducing gene-silencing activity and cell viability, suggesting that ODAGal4 is an efficient stabiliser of siRNAs relative to the other polycation complexes. A plausible explanation for the high efficiency of ODAGal4 for siRNA stabilisation is that the binding selectivity of ODAGal4 to siRNAs. ODAGal4 preferentially binds to RNA duplexes but not to DNA duplexes nor to single-stranded DNAs because the structure of ODAGal4 fits into the major groove of A-form RNA helix but not into that of B-form DNA helix33. We therefore suggest that the binding of ODAGal4 to RNA duplexes is owing to the structural property of ODAGal4 and also to the ionic interaction between the amino groups of ODAGal4 and the nucleotide backbone of the RNA duplexes. In contrast, the other polycations cannot discriminate between RNA and DNA duplex structures and thereby bind to both RNA and DNA nucleotides through the ionic interaction65. Given the low binding specificity, the polycation complexes require the higher N/P ratios for siRNA stabilisation, while ODAGal4 exerts effective and selective stabilisation of siRNAs with the lower N/P ratio. In addition, we have shown in the present study that the efficacy of ODAGal4 for siRNA stabilisation is further amplified by phosphorothioate substitution of siRNAs. We thus suggest that ODAGal4 has a potential advantage as improving siRNA stability and will be a promising tool for reducing total dose and frequency of administration of siRNA-based agents in future applications.It has been reported that various gene delivery systems which consist of cationic polymers  and nucleic acids have been developed to stabilise oligonucleotides for gene therapyAn important goal of future studies will be application of ODAGal4 for siRNA-based drugs. ODAGal4 improved the biological stability of all siRNAs tested in our experiments, strongly suggesting that its effect is independent of siRNA sequence because it does not bind to nucleobases of the RNA duplex. Moreover, as mentioned above, ODAGal4 did not interrupt the gene-silencing activity of any siRNA tested. We thus emphasise that ODAGal4 has great potential for siRNA stabilisation, being widely applicable to various siRNA-based agents.In conclusion, we have demonstrated that the combinations of ODAGal4 and phosphorothioate linkages in RNA duplexes vastly improves siRNA stability, with effects of stabilisation superior to those of other chemical modifications and without compromising gene-silencing activity. Although in vivo studies to confirm and expand our findings are needed, the present results nonetheless provide new insight into development of siRNA-based drugs.33. Sense and antisense single-stranded RNAs were synthesised by Gene Design (Osaka) and Greiner Japan (Tokyo), and annealed in 30 mM HEPES–KOH, pH 7.4, 100 mM CH3COOK and 2 mM Mg(CH3COO)2. The sequences of double-stranded RNA 21mers used are listed in Supplementary Tables ODAGal4, ODAGlc4, ODAMan4 and ODGGal3 were synthesised as described previouslyODAGal4 (20 pmol) and double-stranded RNA oligonucleotides (5 pmol) or single-stranded RNAs (5 pmol) were mixed and then incubated at 37 °C in 10% mouse serum (Sigma-Aldrich) for various time periods. After incubation, the reaction was stopped by the addition of recombinant RNase inhibitor (10 U) (Takara Bio) and EXELDYE 6 × DNA Loading Dye (SMOBIO). In some experiments, siRNAs were digested by 7.5 µg/ml of RNase A (Thermo Fisher Scientific) in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl. The digested RNAs were then separated by a SuperSep DNA 15% native polyacrylamide gel  in 25 mM Tris and 192 mM glycine, stained with SYBR Green II (Takara Bio) in 0.5 × Tris borate EDTA buffer, and visualised with an LAS-4000 image analyser (FUJIFILM). The fluorescence intensity of the remaining RNAs was quantified by using ImageJ software , and the half-lives of RNAs in serum were calculated by using an exponential curve-fitting algorithm in DeltaGraph (Red Rock software).33. In brief, annealed double-stranded RNAs or single-stranded RNAs (2.5 µM) were mixed with ODAGal4 (0–10 µM) in 10 mM NaH2PO4–Na2HPO4 buffer, pH 7.0, containing 100 mM NaCl. UV absorbance of the RNA/ODAGal4 complexes was monitored at 260 and 320 nm with a UV-1650 PC spectrophotometer (SHIMADZU) over a temperature gradient of 0.5 °C/min from 20 to 95 °C. All the UV absorbance values at 260 nm were baseline corrected by subtracting the readings at 320 nm. The thermal melting curves were obtained by plotting the corrected UV absorbance (the A260 reading—the A320 reading) against temperature. The vertical axis was normalised by the value at 20 °C. The Tm value was determined from the peak value of the first derivative of the thermal melting curve.Absorbance versus temperature profiles were assessed as described previously66 and Bifold67 with default settings.RNA secondary structure prediction was conducted by using MaxExpect68. In brief, each annealed RNA (1.5 µM) was prepared in 10 mM NaH2PO4-Na2HPO4 buffer, pH 7.0, containing 100 mM NaCl (250 µL), and the CD spectrum was recorded with a J-820 CD spectropolarimeter (JASCO) at a wavelength of 200 to 320 nm at 37 °C. After measurement, 0.1 mM ODAGal4 (15 µL) was added to the sample solution, and the CD spectrum was recorded again. The instrument settings were resolution, 0.1 nm; sensitivity, 5 mdeg; response, 1 s; speed, 100 nm/min; accumulation, 20.CD spectroscopy was conducted as described previously33. In brief, 5′-FAM-labelled RNA duplexes (100 nM) were titrated with 0–700 nM ODAGal4 in 10 mM NaH2PO4–Na2HPO4 buffer, pH 7.0, containing 100 mM of NaCl and 0.02% Tween 20 at 20 °C. The change in fluorescence anisotropy was monitored with a FP-6500 spectrofluorometer (JASCO) by averaging three measurements. The instrument settings were Ex/Em = 490/520 nm; response, 2 s; bandwidth (Ex), 5 nm; bandwidth (Em), 5 nm; PMT voltage, 430 V; number of cycles, 4. The dissociation constant of ODAGal4 and each RNA duplex was calculated by the method of Wang et al.69Fluorescence anisotropy was conducted as described previously4 cells) were cultured for a day in Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) FBS in 48-well plates. For the transfection complex, RNA (2.5 pmol) was mixed with ODAGal4 (10 pmol) at room temperature, and then Lipofectamine RNAiMAX (0.75 µg) (Thermo Fisher Scientific) was added. In some experiments, RNA (2.5 pmol) with ODAGal4 (10 pmol) was incubated at 37 °C in 10% mouse serum for 0 to 72 h, and then mixed with Lipofectamine RNAiMAX (0.75 µg). The cell culture medium was replaced with 250 µL of Opti-MEM medium (Thermo Fisher Scientific), and the RNA/ODAGal4/Lipofectamine complex was added to the cells. After 4 h, the transfection complex was removed, and the cells were cultured in DMEM with 10% FBS for 2 days before gene expression analysis. The negative control siRNA was 5′-GUACCGCACGUCAUUCGUAUC-3′ (sense) and 5′-UACGAAUGACGUGCGGUACGU-3′ (antisense).HeLa cells and Hep3B cells were obtained from Japanese Collection of Research Bioresources Cell Bank and American Type Culture Collection, respectively. Cells , and reverse-transcribed by using PrimeScript RT Master Mix (Takara Bio) in accordance with the manufacturer’s protocol. Quantitative PCR analysis was performed with TB Green Premix Ex Taq II (Takara Bio) and a LightCycler 480 (Roche). The relative expression level of the mRNAs for B2M and HPRT1 genes was calculated by the ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase as an internal control. The oligonucleotide primers for detection were designed by using ProbeFinder software (Roche): B2M, 5′-TTCTGGCCTGGAGGCTATC-3′ and 5′-TCAGGAAATTTGACTTTCCATTC-3′; glyceraldehyde 3-phosphate dehydrogenase, 5′-AGCCACATCGCTCAGACAC-3′ and 5′-GCCCAATACGACCAAATCC-3′; HPRT1, 5′-TGACCTTGATTTATTTTGCATACC-3′ and 5′-CGAGCAAGACGTTCAGTCCT-3′. The RHeLa cells were transfected by RNA (2.5 pmol) with or without ODAGal4 (10 pmol) and cultured for 2 days at the same condition as described above. Cell viability was determined by the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) method by using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega) in accordance with the manufacturer’s protocol.t test. In the figures, statistical significance is denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.Data were expressed as the mean ± SE. Statistical significance was assessed by unpaired Student’s Supplementary Information.


It is a lipid-soluble antioxidant and a very effective quencher of reactive oxygen species (ROS) to reduce the oxidative stress. In contrast to vitamin A, β-carotene is not toxic even consumed in higher amount when it is delivered from natural plant products. Recently, we found that β-carotene acts as a potential antioxidant in the oocyte to improve its quality. Even though many studies have been reported that β-carotene has the beneficial contribution to the ovarian development and steroidogenesis, it is unknown the effects of β-carotene on the spermatogenesis. This investigation aimed to explore the hypothesis that β-carotene could improve spermatogenesis and the underlying mechanism. And we found that β-carotene rescued busulfan disrupted spermatogenesis in mouse with the increase in the sperm concentration and motility. β-carotene improved the expression of genes/proteins important for spermatogenesis, such as VASA, DAZL, SYCP3, PGK2. Moreover, β-carotene elevated the testicular antioxidant capability by the elevation of the antioxidant glutathione and antioxidant enzymes SOD, GPX1, catalase levels. In conclusion, β-carotene may be applied for the infertile couples by the improvement of spermatogenesis, since, worldly many couples are infertile due to the idiopathic failed gametogenesis (spermatogenesis). Infertility is a growing issue worldwide which now impacts 10%–15% of all couples with half of the cases being attributed to male factors . Epidemi40H56) is one of the major dietary carotenoids  in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . Detail Mice werThree-week-old ICR male mice were given a single injection of busulfan [40 mg/kg body weight (BW)] . We woulSpermatozoa motility and concentration were assessed by a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines . The spePlasma testosterone (T) and oestrogen (E) levels were quantified by mouse testosterone Elisa Kit and mouse estradiol Elisa Kit, respectively, from Nanjing Jiancheng Bioengineering Institute  as reported in our early publication .Plasma aspartate aminotransferase  and alanine aminotransferase  were measured by the kits from Nanjing Jiancheng Bioengineering Institute  as reported in our previous publication .The activity of catalase (Cat #: A007-1-1), GPX1 (Cat #: A005), SOD (Cat #: A001-3), T-AOC (Cat #: A015-2-1) in mouse testis were detected by the kits from Nanjing Jiancheng Bioengineering Institute  as reported in our previous publication .The testis levels of GSH/GSSG (Cat #: S0053) were detected by the kits from Beyotime Bioengineering Institute  as reported in our previous publication .Western blotting analysis was followed the procedure reported in our previous publications . The souThe procedure for immunofluorescent staining was reported in our recent publications . Supplemp < 0.05.The data were determined by SPSS statistical software  with one-way analysis of variance (ANOVA) following by LSD multiple comparison test. All groups were compared with each other for every parameter. The data were shown as the mean ± SEM. Statistically significant was based on As shown in Meiosis plays important roles in the controlling germ cell development to haploid and maintaining genetic diversity. It has been reported that the abnormal meiosis will induce aneuploidy formation, even cause infertility, miscarriage or even birth defect. In current investigation, busulfan disrupted the meiosis process during the spermatogenesis with the alteration in the percentage of cells at different stages . The perIn order to confirm β-carotene improving spermatogenesis and search for the mode of action of it in rescuing spermatogenesis, the gene and protein expression of important factors in spermatogenesis was determined. We did Q-RT-PCR for a lot of genes important for spermatogenesis, and we found that the expression of eight genes was increased by B + β-caro 10 group compared to Busulfan alone, while just PIWIL1 was dramatically increased by β-caro 10 compared to control . At the To explore the deep mechanisms of β-carotene improving spermatogenesis, the anti-oxidant enzymes and glutathione (GSH) level in testes were determined. We and other groups have found that β-carotene can increase the anti-oxidant capability to benefit the function of the biological systems . GSH is It has been reported that the carotenoids in fruits play very important roles to prevent diseases and maintain body functions. Moreover, many investigations suggested β-carotene, lycopene, lutein, zeaxanthin and other carotenoids possess lots of health benefits. The blood concentration of β-carotene is about 2.54–3.3 μg/ml. It has been applied in many animal investigations such as cow, goat, rabbit, and sow . In currβ-carotene improved busulfan disrupted meiosis process. Busulfan decreased the percentage of cells in late phases, while β-carotene restored the percentage of these cells to the control level. Meiosis is an important process to keep germ cell development. And the abnormal meiosis may upset the haploid formation which may lead to the infertility, miscarriage, and so on . BusulfaIt has been reported busulfan increased intracellular oxidative stress . And β-cIn summary, β-carotene rescued busulfan disrupted spermatogenesis by improving meiosis process, the expression of important genes/proteins in spermatogenesis process, testicular antioxidant capability. These beneficial advantages of β-carotene may be applied to ameliorate male reproduction for the patients who are under busulfan or other cancer-drug treatments. As natural product, β-carotene may be applied for the infertile couples especially cancer-drug treated patients by the improvement of spermatogenesis, since, worldwide many couples are infertile due to the idiopathic failed gametogenesis (spermatogenesis) .