Downstream Coding Sequences Research Articles

Computational identification of biologically functional non-hairpin GC-helices in human Argonaute mRNA

BackgroundPerfectly formed duplex elements in RNA occur within folding units, often as a part of hairpin motifs which can be reliably predicted by various RNA folding algorithms. Double helices with consecutive Watson-Crick base-pairing may also be formed between distant RNA segments thereby facilitating long-range interactions of long-chain RNA that may be biologically functional. Here we addressed the potential formation of RNA duplex motifs by long-range RNA-RNA interactions of distantly located matching sequence elements of a single long-chain RNA.ResultsWe generated a Python-based software tool that identifies consecutive RNA duplex elements at any given length and nucleotide content formed by distant sequences. The software tool, dubbed RNAslider, is built on the theoretical RNA structure prediction algorithm Mfold. Source code and sample data sets are available on demand. We found that a small ratio of human genes including the Argonaute (Ago)-like gene family encode mRNAs containing highly GC-rich non-hairpin duplex elements (GC-helix) of equal to or more than 8 base pairs in length and we provide experimental evidence for their biological significance.ConclusionGC-helices are observed preferentially within the 5′-region of mRNAs in an evolutionarily conserved fashion indicating their potential biological role. This view is supported experimentally by post-transcriptional regulation of gene expression of a fusion transcript containing 5′-sequences of human mRNAAgo2 harbouring GC-helices and down-stream coding sequences of Renilla luciferase.

The Stringency of Start Codon Selection in the Filamentous Fungus Neurospora crassa

In eukaryotic cells initiation may occur from near-cognate codons that differ from AUG by a single nucleotide. The stringency of start codon selection impacts the efficiency of initiation at near-cognate codons and the efficiency of initiation at AUG codons in different contexts. We used a codon-optimized firefly luciferase reporter initiated with AUG or each of the nine near-cognate codons in preferred context to examine the stringency of start codon selection in the model filamentous fungus Neurospora crassa. In vivo results indicated that the hierarchy of initiation at start codons in N. crassa (AUG ≫ CUG > GUG > ACG > AUA ≈ UUG > AUU > AUC) is similar to that in human cells. Similar results were obtained by translating mRNAs in a homologous N. crassa in vitro translation system or in rabbit reticulocyte lysate. We next examined the efficiency of initiation at AUG, CUG, and UUG codons in different contexts in vitro. The preferred context was more important for efficient initiation from near-cognate codons than from AUG. These studies demonstrated that near-cognate codons are used for initiation in N. crassa. Such events could provide additional coding capacity or have regulatory functions. Analyses of the 5'-leader regions in the N. crassa transcriptome revealed examples of highly conserved near-cognate codons in preferred contexts that could extend the N termini of the predicted polypeptides.

Selective Constraint on the Upstream Open Reading Frames That Overlap with Coding Sequences in Animals

Upstream open reading frames (uORFs) are translational regulatory elements located in 5′ untranslated regions. They can significantly repress the translation of the downstream coding sequences (CDS), and participate in the spatio-temporal regulations of protein translation. Notwithstanding this biological significance, the selective constraint on uORFs remains underexplored. Particularly, the uORFs that partially overlap with CDS with a different reading frame (overlapping uORFs, or “VuORFs”) may lead to strong translational inhibition or N-terminal truncation of the peptides encoded by the affected CDS. By analyzing VuORF-containing transcripts (designated as “VuORF transcripts”) in human, mouse, and fruit fly, we demonstrate that VuORFs are in general slightly deleterious - the proportion of genes that encode at least one VuORF transcript is significantly smaller than expected in all of the three examined species. In addition, this proportion is significantly smaller in fruit fly than in mammals, indicating a higher efficiency of removing VuORFs in the former species because of its larger effective population size. Furthermore, the deleterious effect of a VuORF depends on the sequence context of its start codon (VuAUG). VuORFs with an optimal VuAUG context are more strongly disfavored than those with a suboptimal context in all of the three examined species. And the propensity to remove optimal-context VuAUGs is stronger in fruit fly than in mammals. Intriguingly, however, the currently observable optimal-context VuAUGs (but not suboptimal-context VuAUGs) are more conserved than expected. These observations suggest that the regulatory functions of VuORFs may have been gained fortuitously in organisms with a small effective population size because the slightly deleterious effect of these elements can be better tolerated in these organisms, thus allowing opportunities for the development of novel biological functions. Nevertheless, once the functions of VuORFs were established, they became subject to negative selection.

Direct and specific chemical control of eukaryotic translation with a synthetic RNA–protein interaction

Sequence-specific RNA–protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA–protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5′-untranslated region (5′-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5′-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA–TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

ERG protein expression in prostate and vascular tumors: A potential for ERG-based stratification.

e15171 Background: Frequent overexpression of ERG in prostate tumors is a result of prevalent gene fusions involving upstream sequences of androgen regulated genes (predominantly TMPRSS2) and downstream coding sequences of nuclear transcription factors within the ETS gene family (primarily ERG). Despite numerous reports of ERG alterations at the transcript and genome levels, the ERG oncoprotein remains to be defined. Using a highly specific ERG monoclonal antibody, we have recently developed a comprehensive insight into ERG expression in various cell types of benign and malignant prostate glands. Further, we have analyzed ERG protein expression in human tumors of diverse origins. Methods: Using whole-mount prostate sections from 150 prostatectomy specimens, ERG protein expression was analyzed in PIN, tumor foci, benign glands and other cell types in prostate. In randomly selected cases ERG protein expression was correlated with ERG fusion status. A broad range of vascular endothelial (n=250), other mesenchymal (n=973), and epithelial tumors (n=657) were examined. Results: CPDR ERG-MAb showed striking specificity for detecting prostate tumor cells (>99.9%). Specimens from 65% patients had one or more ERG positive tumor focus. Examination of ERG positive PINs and ERG positive tumors within the same whole-mount sections revealed a 97% concordance. In normal adult tissues, including human and mouse prostate, ERG was restricted to endothelial cells and a subset of bone marrow precursors. ERG was expressed in virtually all vascular tumors and absent in all epithelial tumors except prostate tumors. Conclusions: Taken together, the homogeneous and strong ERG expression in individual tumors and the remarkable correlation between ERG positive PIN lesions and tumors suggests a role for ERG in the pre-invasive to invasive transition of prostate cancer cells. The demonstrated high specificity of CPDR ERG-MAb has potential to provide new opportunities in prostate cancer diagnosis and stratification. Further, ERG is a highly specific new marker for benign and malignant vascular tumors.

The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences

Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.

Differential Expression of Novel Adiponectin Receptor-1 Transcripts in Skeletal Muscle of Subjects With Normal Glucose Tolerance and Type 2 Diabetes

OBJECTIVEAdiponectin receptor-1 (AdipoR1) expression in skeletal muscle has been suggested to play an important role in insulin resistance and diabetes. We aimed at evaluating the presence of novel AdiopR1 splice variants in human muscle and their regulation under physiological and pathophysiological states.RESEARCH DESIGN AND METHODSAdipoR1 5′UTR mRNA transcripts, predicted from bioinformatics data, were evaluated in fetal and adult human tissues. Expression and function of the identified transcripts were assessed in cultured human skeletal muscle cells and in muscle biopsies obtained from individuals with normal glucose tolerance (NGT) and type 2 diabetes (n = 49).RESULTSScreening of potential AdipoR1 5′UTR splice variants revealed a novel highly abundant muscle transcript (R1T3) in addition to the previously described transcript (R1T1). Unlike R1T1, R1T3 expression was significantly increased during fetal development and myogenesis, paralleled with increased AdipoR1 protein expression. The 5′UTR of R1T3 was found to contain upstream open reading frames that repress translation of downstream coding sequences. Conversely, AdipoR1 3′UTR was associated with enhanced translation efficiency during myoblast-myotube differentiation. A marked reduction in muscle expression of R1T3, R1T1, and R1T3-to-R1T1 ratio was observed in individuals with type 2 diabetes compared with expression levels of NGT subjects, paralleled with decreased expression of the differentiation marker myogenin. Among NGT subjects, R1T3 expression was positively correlated with insulin sensitivity.CONCLUSIONSThese results indicate that AdipoR1 receptor expression in human skeletal muscle is subjected to posttranscriptional regulation, including alternative splicing and translational control. These mechanisms play an important role during myogenesis and may be important for whole-body insulin sensitivity.

Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [(3)H]estradiol-beta-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

RNA switches regulate initiation of translation in bacteria

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.

Natural Variability in S -Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis Exhibit Differential Sensitivity to SAM In Vivo and In Vitro

Riboswitches are regulatory systems in which changes in structural elements in the 5' region of the nascent RNA transcript (the "leader region") control expression of the downstream coding sequence in response to a regulatory signal in the absence of a trans-acting protein factor. The S-box riboswitch, found primarily in low-G+C gram-positive bacteria, is the paradigm for riboswitches that sense S-adenosylmethionine (SAM). Genes in the S-box family are involved in methionine metabolism, and their expression is induced in response to starvation for methionine. S-box genes exhibit conserved primary sequence and secondary structural elements in their leader regions. We previously demonstrated that SAM binds directly to S-box leader RNA, causing a structural rearrangement that results in premature termination of transcription at S-box leader region terminators. S-box genes have a variety of physiological roles, and natural variability in S-box structure and regulatory response could provide additional insight into the role of conserved S-box leader elements in SAM-directed transcription termination. In the current study, in vivo and in vitro assays were employed to analyze the differential regulation of S-box genes in response to SAM. A wide range of responses to SAM were observed for the 11 S-box-regulated transcriptional units in Bacillus subtilis, demonstrating that S-box riboswitches can be calibrated to different physiological requirements.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Dual Modes of Natural Selection on Upstream Open Reading Frames

Upstream open reading frames (uORFs) are common features of eukaryotic genes, occurring in 10%-25% of 5' leader sequences. Upstream ORFs that have been subjected to experimental analysis have been generally found to decrease translational efficiency of the downstream coding sequence. Previous investigations of uORFs in mammals and yeast have detected uORFs conserved over long evolutionary distances, prompting speculation about the nature and cause of the natural selection underlying such conservation. We have analyzed uORFs in the basidiomycetous fungal pathogen Cryptococcus neoformans to discern the properties of this purifying selection. We find that uORFs in the Cryptococcus species complex are conserved at twice the expected rate, and we report 122 uORFs that are conserved among all four sequenced Cryptococcus strains. A significantly greater proportion of uORF losses occur via direct mutation to the uORF start codon than expected. This observation suggests that mutational disruption of a uORF that leaves the start codon intact may be selectively disadvantageous, perhaps because of the risk of premature translation initiation. Accounting for this constrained mode of loss and comparing the relative conservation of uORFs between the 5' leader and control sequences enables us to calculate that at least a third of uORFs may be conserved for their effects on translational efficiency. The remaining fraction may be conserved either by chance or as a result of selective pressure to prevent premature translation initiation from the uORF start codon. We find that the majority of conserved uORFs do not exhibit codon usage bias or conservation at the amino acid level, and therefore they do not likely encode bioactive peptides. Our analysis suggests that uORFs are an important and underappreciated mechanism of post-transcriptional gene regulation in eukaryotes.

Binding Mode of the First Aminoacyl-tRNA in Translation Initiation Mediated by Plautia stali Intestine Virus Internal Ribosome Entry Site

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

Identification of a Mutation in the Bacillus subtilis S -Adenosylmethionine Synthetase Gene That Results in Derepression of S-Box Gene Expression

Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5' region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

Sensing Metabolic Signals with Nascent RNA Transcripts: The T Box and S Box Riboswitches as Paradigms

Recent studies in a variety of bacterial systems have revealed a number of regulatory systems in which the 5' region of a gene plays a key role in regulation of the downstream coding sequences. These RNA regions act in cis to determine if the full-length transcript will be synthesized or if the coding sequence(s) will be translated. Each class of system includes an RNA element whose structure is modulated in response to a specific regulatory signal, and the signals measured can include small molecules, small RNAs (including tRNA), and physical parameters such as temperature. Multiple sets of genes can be regulated by a particular mechanism, and multiple systems of this type, each of which responds to a specific signal, can be present in a single organism. In addition, different classes of RNA elements can be found that respond to a particular signal, indicating the existence of multiple alternate solutions to the same regulatory problem. The T box and S box systems, which respond to uncharged tRNA and S-adenosylmethionine (SAM), respectively, provide paradigms of two systems of this type.

From Ribosome to Riboswitch: Control of Gene Expression in Bacteria by RNA Structural Rearrangements

ABSTRACTStructural elements in the 5′ region of a bacterial mRNA can have major effects on expression of downstream coding sequences. Folding of the nascent RNA into the helix of an intrinsic transcriptional terminator results in premature termination of transcription and in failure to synthesize the full-length transcript. Structure in the translation initiation region of an mRNA blocks access of the translation initiation complex to the ribosome binding site, thereby preventing protein synthesis. RNA structures can also affect the stability of an RNA by altering sensitivity to ribonucleases. A wide variety of mechanisms have been uncovered in which changes in mRNA structure in response to a regulatory signal are used to modulate gene expression in bacteria. These systems allow the cell to recognize an impressive array of signals, and to monitor those signals in many different ways.

Effect of 5′‐proximal elements on decay of a model mRNA in Bacillus subtilis

Previous work showed that a 42-nucleotide sequence from an SP82 bacteriophage early RNA functions as a 5' mRNA stabilizer in Bacillus subtilis. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of decay of a model mRNA with alterations at the 5'-end was used to elucidate the mechanism of SP82-mediated stability. A predicted 5'-terminal stem-loop structure was essential for stabilization. Increasing the strength of the 5'-terminal structure above a minimum level did not result in increased stability. A thorough analysis of the context in which the stabilizing structure occurred included the effects of distance from 5'-end, translation of downstream coding sequence, and distance between the secondary structure and the ribosome binding site. Our data are consistent with the dominant mRNA decay pathway in B. subtilis being 5'-end dependent.

Expression of AtMHX, an Arabidopsis vacuolar metal transporter, is repressed by the 5' untranslated region of its gene

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.

A naturally occurring non-coding fusion transcript derived from scorpion venom gland: implication for the regulation of scorpion toxin gene expression

Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced. However, their expression regulation mechanism is not yet known at present. During screening of a cDNA library prepared from venom glands of the scorpion Buthus martensii Karsch, we isolated a natural fusion cDNA composed of the 5′-untranslated region (UTR) and upstream coding sequence of a long-chain toxin transcript and the downstream coding sequence and 3′-UTR of a short-chain toxin transcript. The junction site is just the overlapping region of 11 nucleotides (GGCAAGGAAAT) between the two wild transcripts, and thus leads to the formation of an early stop codon, which will cause premature translation. Based on the above observations, combined with the genomic data, we proposed a characteristic regulation mechanism of scorpion toxin genes, in which trans-splicing and nonsense mediated mRNA decay are involved.