Leader RNA facilitates snakehead vesiculovirus (SHVV) replication by interacting with CSDE1 and hnRNP A3.

Leader RNAs are viral small non-coding RNAs that has been proved to play important roles in viral replication. Snakehead vesiculovirus (SHVV) is an aquatic virus that has caused huge economic loss in Chinese snakehead fish aquaculture industry. It has been proved that SHVV would generate leader RNA during the process of infection, and leader RNA could interact with viral nucleoprotein to promote viral replication. In this study, we identified that leader RNA could also interact with cellular protein Cold Shock Domain containing E1 (CSDE1) and heterogeneous nuclear ribonucleoproteins A3 (hnRNP A3). Further investigation reveals that overexpression of CSDE1 and hnRNP A3 facilitated SHVV replication. Downregulation of CSDE1 and hnRNP A3 by siRNA inhibited SHVV replication. This study provided a new sight into understand the mechanism of SHVV replication, and a potential anti-SHVV target for drug research.

Snakehead vesiculovirus (SHVV) leader RNA interacts with host antiviral factors RPS8 and L13a and promotes virus replication.

To evade host antiviral response, viruses have evolved to take advantage of their noncoding RNAs (ncRNAs). Snakehead vesiculovirus (SHVV), a newly isolated fish rhabdovirus from diseased hybrid snakehead, has caused high mortality to the cultured snakehead fish during the past years in China. However, little is known about the mechanisms of its pathogenicity. Our study revealed that overexpression of the 30-nt leader RNA promoted SHVV replication. RNA-protein binding investigation revealed that SHVV leader RNA could interact with host 40S ribosomal protein S8 (RPS8) and 60S ribosomal protein L13a (L13a). Furthermore, we found that SHVV infection upregulated RPS8 and L13a, and in turn, overexpression of RPS8 or L13a inhibited, while knockdown of RPS8 or L13a promoted, SHVV replication, suggesting that RPS8 and L13a acted as host antiviral factors in response to SHVV infection. In addition, our study revealed that RPS8- or L13a-mediated inhibition of SHVV replication could be restored by co-transfection with leader RNA, suggesting that the interaction between leader RNA and RPS8 or L13a might affect the anti-SHVV effects of RPS8 and L13a. Taken together, these results suggest that SHVV leader RNA can interact with the host antiviral factors RPS8 and L13a, and promote SHVV replication. This study provides a better understanding of the molecular mechanism of the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

Leader RNA regulates snakehead vesiculovirus replication via interacting with viral nucleoprotein.

Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.

A trans-acting leader RNA from a Salmonella virulence gene.

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

The response of trypanosomes and other eukaryotes to ER stress and the spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei.

The unfolded protein response (UPR) is induced when the quality control machinery of the cell is overloaded with unfolded proteins or when one of the functions of the endoplasmic reticulum (ER) is perturbed. Here, I describe UPR in yeast and mammals, and compare it to what we know about pathogenic fungi and the parasitic protozoans from the order kinetoplastida, focusing on the novel pathway the spliced leader silencing (SLS) in Trypanosoma brucei. Trypanosomes lack conventional transcription regulation, and thus, lack most of the UPR machinery present in other eukaryotes. Trypanosome genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon known as the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Under severe ER stress, T. brucei elicits the SLS pathway. In SLS, the transcription of the SL RNA gene is extinguished, and the entire transcription complex dissociates from the SL RNA promoter. Induction of SLS is mediated by an ER-associated kinase (PK3) that migrates to the nucleus, where it phosphorylates the TATA-binding protein (TRF4), leading shut-off of SL RNA transcription. As a result, trans-splicing is inhibited and the parasites activate a programmed cell death (PCD) pathway. Despite the ability to sense the ER stress, the different eukaryotes, especially unicellular parasites and pathogenic fungi, developed a variety of unique and different ways to sense and adjust to this stress in a manner different from their host.

Euglena Transcript Processing.

RNA transcript processing is an important stage in the gene expression pathway of all organisms and is subject to various mechanisms of control that influence the final levels of gene products. RNA processing involves events such as nuclease-mediated cleavage, removal of intervening sequences referred to as introns and modifications to RNA structure (nucleoside modification and editing). In Euglena, RNA transcript processing was initially examined in chloroplasts because of historical interest in the secondary endosymbiotic origin of this organelle in this organism. More recent efforts to examine mitochondrial genome structure and RNA maturation have been stimulated by the discovery of unusual processing pathways in other Euglenozoans such as kinetoplastids and diplonemids. Eukaryotes containing large genomes are now known to typically contain large collections of introns and regulatory RNAs involved in RNA processing events, and Euglena gracilis in particular has a relatively large genome for a protist. Studies examining the structure of nuclear genes and the mechanisms involved in nuclear RNA processing have revealed that indeed Euglena contains large numbers of introns in the limited set of genes so far examined and also possesses large numbers of specific classes of regulatory and processing RNAs, such as small nucleolar RNAs (snoRNAs). Most interestingly, these studies have also revealed that Euglena possesses novel processing pathways generating highly fragmented cytosolic ribosomal RNAs and subunits and non-conventional intron classes removed by unknown splicing mechanisms. This unexpected diversity in RNA processing pathways emphasizes the importance of identifying the components involved in these processing mechanisms and their evolutionary emergence in Euglena species.

A diversified and segregated mRNA spliced-leader system in the parasitic Perkinsozoa.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5' exon, giving different mRNAs identical 5' sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

N-protein-RNA interaction is a drug target in a negative strand RNA virus.

The negative strand RNA virus family contains many human pathogens. Finding new antiviral drug targets against this class of human pathogens is one of the significant healthcare needs. Nucleocapsid proteins of negative strand RNA viruses wrap the viral genomic RNA and play essential roles in gene transcription and genome replication. Chandipura virus, a member of the Rhabdoviridae family, has a negative strand RNA genome. In addition to wrapping the genomic RNA, its nucleocapsid protein interacts with the positive strand leader RNA and plays a vital role in the virus life-cycle. We have designed a peptide, based on prior knowledge and demonstrated that the peptide is capable of binding specifically to the positive strand leader RNA. When the peptide was transported inside the cell, it inhibited viral growth with IC50 values in the low micromolar range. Given the widespread occurrence of leader RNAs in negative strand RNA viruses and its interaction with the nucleocapsid protein, it is likely that this interaction could be a valid drug target for other negative strand RNA viruses.

The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3.

In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5' exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments. IMPORTANCE In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. We also report on the autophosphorylation of PK3 during SLS induction. This study has implications for our understanding of how trypanosomes keep the homeostasis between the ER and the mitochondria and suggests that PK3 may participate in the connection between these two organelles. The pathway, when induced, leads to the suicide of these parasites, and its induction offers a potential novel drug target against these parasites.

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication.

Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication.

A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.

Host-specificity of Monoxenous Trypanosomatids: Statistical Analysis of the Distribution and Transmission Patterns of the Parasites from Neotropical Heteroptera.

Host-parasite relationships and parasite biodiversity have been the center of attention for many years; however the primary data obtained from large-scale studies remain scarce. Our long term investigations of trypanosomatid (Euglenozoa: Kinetoplastea) biodiversity from Neotropical Heteroptera have yielded almost one hundred typing units (TU) of trypanosomatids from one hundred twenty host species. Half of the parasites' TUs were documented in a single host species only but the rest were found parasitizing two to nine species of hosts, with logarithmic distribution best describing the observed distribution of parasites among hosts. Different host superfamilies did not show significant differences in numbers of trypanosomatid TUs they carry, with exception of Pyrrhocoroidea which showed higher parasite richness than any other group tested. Predatory reduviids shared significantly larger numbers of parasite TUs with phytophagous mirids and coreids than the numbers shared between any other groups. These results show that the specificity of trypanosomatid-heteropteran associations is not very strict: parasites seem to be transmissible between different host groups within the same niche and predatory hosts may acquire parasites from their prey.

Transcriptional mapping of the messenger and leader RNAs of orchid fleck virus, a bisegmented negative-strand RNA virus.

The transcriptional strategy of orchid fleck virus (OFV), which has a two-segmented negative-strand RNA genome and resembles plant nucleorhabdoviruses, remains unexplored. In this study, the transcripts of six genes encoded by OFV RNA1 and RNA2 in the poly(A)-enriched RNA fraction from infected plants were molecularly characterized. All of the OFV mRNAs were initiated at a start sequence 3'-UU-5' with one to three non-viral adenine nucleotides which were added at the 5' end of each mRNA, whereas their 3' termini ended with a 5'-AUUUAAA(U/G)AAAA(A)n-3' sequence. We also identified the presence of polyadenylated short transcripts derived from the 3'-terminal leader regions of both genomic and antigenomic strands, providing the first example of plus- and minus-strand leader RNAs in a segmented minus-strand RNA virus. The similarity in the transcriptional strategy between this bipartite OFV and monopartite rhabdoviruses, especially nucleorhabdoviruses (family Rhabdoviridae) is additional support for their close relationship.

Molecular cloning and characterization of SL3: a stem cell-specific SL RNA from the planarian Schmidtea mediterranea.

Spliced leader (SL) trans-splicing is a biological phenomenon, common among many metazoan taxa, consisting in the transfer of a short leader sequence from a small SL RNA to the 5' end of a subset of pre-mRNAs. While knowledge of the biochemical mechanisms driving this process has accumulated over the years, the functional consequences of such post-transcriptional event at the organismal level remain unclear. In addition, the fact that functional analyses have been undertaken mainly in trypanosomes and nematodes leaves a somehow fragmented picture of the possible biological significance and evolution of SL trans-splicing in eukaryotes. Here, we analyzed the spatial expression of SL RNAs in the planarian flatworm Schmidtea mediterranea, with the goal of identifying novel developmental paradigms for the study of trans-splicing in metazoans. Besides the previously identified SL1 and SL2, S. mediterranea expresses a third SL RNA described here as SL3. While, SL1 and SL2 are collectively expressed in a broad range of planarian cell types, SL3 is highly enriched in a subset of the planarian stem cells engaged in regenerative responses. Our findings provide new opportunities to study how trans-splicing may regulate the phenotype of a cell.

Diversity of trypanosomatids (Kinetoplastea: Trypanosomatidae) parasitizing fleas (Insecta: Siphonaptera) and description of a new genus Blechomonas gen. n.

To further investigate the diversity of Trypanosomatidae we have examined the species present within the flea (Siphonaptera) population in the Czech Republic. Out of 1549 fleas, 239 were found to be infected by trypanosomatids. Axenic cultures were established from 90 infected specimens and 29 of them were further characterized. Molecular phylogenetic analysis of the SL RNA, gGAPDH, and SSU rRNA genes revealed a striking diversity within this group and analyzed isolates were classified into 16 Typing units (TUs) of which 15 typified new species. In addition to one Trypanosoma species, two TUs grouped within the sub-family Leishmaniinae, two clustered together with Herpetomonas, wheras 11 TUs formed a novel clade branching off between Trypanosoma spp. and remaining trypanosomatids. We propose to recognize this clade as a new genus Blechomonas and a new subfamily Blechomonadinae, and provide molecular and morphological description of 11 TUs representing this genus. Our finding of such an ancient host-specific group sheds new light at the origin of Trypanosomatidae and the roots of dixenous parasitism. The strict host restriction of Blechomonas to Siphonaptera with adult fleas' dependence on blood meal may reflect passing of parasites from larvae through pupae to adults and implies potential transmission to the warm-blooded vertebrates.

A peptide targeted against phosphoprotein and leader RNA interaction inhibits growth of Chandipura virus -- an emerging rhabdovirus.

The fatal illness caused by Chandipura virus (CHPV), an emerging pathogen, presently lacks any therapeutic option. Previous research suggested that interaction between the virally encoded phosphoprotein (P) and the positive sense leader RNA (le-RNA) may play an important role in the viral lifecycle. In this report, we have identified a ß-sheet/loop motif in the C-terminal domain of the CHPV P protein as essential for this interaction. A synthetic peptide encompassing this motif and spanning a continuous stretch of 36 amino acids (Pep208-243) was found to bind the le-RNA in vitro and inhibit CHPV growth in infected cells. Furthermore, a stretch of three amino acid residues at position 217-219 was identified as essential for this interaction, both in vitro and in infected cells. siRNA knockdown-rescue experiments demonstrated that these three amino acid residues are crucial for the leader RNA binding function of P protein in the CHPV life cycle. Mutations of these three amino acid residues render the peptide completely ineffective against CHPV. Effect of inhibition of phosphoprotein-leader RNA interaction on viral replication was assayed. Peptide Pep208-243 tagged with a cell penetrating peptide was found to inhibit CHPV replication as ascertained by real time RT-PCR. The specific inhibition of viral growth observed using this peptide suggests a new possibility for designing of anti-viral agents against Mononegavirale group of human viruses.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Efficient Depletion of Multiple SARS-CoV mRNAs by a Single Small Interfering RNA Targeting The Leader Sequence / 生物化学与生物物理进展

Small interfering RNAs (siRNAs) can efficiently inhibit gene expression by sequence-specific RNA interference (RNAi). A common 5' leader sequence exists in the genomic RNA and all subgenomic RNAs of SARS-CoV, and is well conserved among various SARS-CoV strains, thus providing a preferable target for RNAi of SARS-CoV replication. Here efficient depletion of the SARS-CoV mRNAs by either a synthetic siRNA or DNA vector-derived short hairpin RNAs (shRNAs) targeting the leader sequence in mammalian cell lines were reported. The siRNA or shRNAs efficiently suppressed the expression of an EGFP reporter gene which contains the leader sequence at the 5' end. Both the siRNA and shRNAs efficiently knocked down the levels of leader-containing transcripts of three SARS-CoV genes encoding the spike protein, membrane protein and nucleocapsid protein were demonstrated. The results suggest that RNAi targeting the leader sequence is a potential efficient strategy for anti-SARS-CoV therapy.