Dicistronic tRNA-mRNA transcripts in grapevine (Vitis vinifera) display

20 Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and 21 amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures can also act 22 as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a 23 common feature in the plant nuclear genome and, in the few cases where polycistronic 24 transcripts have been found, they include the expression of non-coding RNA species such as 25 small nucleolar RNAs and microRNA clusters. It is not known, however, the extent to which 26 dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors 27 contributing to their expression. To address these questions, we analysed tRNA-mRNA 28 dicistronic transcripts in the major horticultural crop grapevine (Vitis vinifera) using a novel 29 pipeline developed to identify dicistronic transcripts from high-throughput RNA sequencing 30 data. We identified dicistronic tRNA-mRNA in grapevine leaf and berry samples from 22 31 commercial vineyards covering six sub-regions of the Barossa wine growing region, Australia. 32 Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming 33 part of 19 dicistronic tRNA-mRNA molecules. The presence and the abundance of dicistronic 34 molecules was tissue and geographic sub-region specific. In leaf tissue, the expression patterns 35 of dicistronic tRNA-mRNAs significantly correlated with tRNA expression, suggesting that 36 transcriptional regulation of their expression might be linked. We also found evidence of 37 evolutionary conservation of dicistronic candidates in grapevine, and previously reported 38 dicistronic transcripts in Arabidopsis, indicating a syntenic genomic arrangement of tRNAs and 39 protein coding genes between species. 40


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Institute & School of Agriculture, Food and Wine, University of Adelaide, Adelaide, Australia 1 1 1 3 8 dicistronic tRNA-mRNA pairs. Conversely, no significant difference in coverage was observed 1 3 9 for tRNA-mRNA pairs deemed non-dicistronic (Supplemental Fig S2). In total, 19 individual tRNA genes, representing 13 isoacceptor families were found to be 1 4 1 dicistronic with the neighbouring protein coding genes, among which, glycine tRNA genes were 1 4 2 the most common. We validated, through RT-PCR, two randomly selected tRNA-mRNA  Table S3). We then analysed the expression of the tRNA genes, the intergenic regions and PCGs forming dicistronic transcripts ( Figure 6). Sub-regional clusters for tRNAs forming part of dicistronic 1 9 0 constructs were similar to those observed for all expressed tRNAs in both tissues ( Figure 6). In intergenic regions and dicistronic PCGs was used rather than tRNA, sub-regional clustering was 1 9 6 tissue and dicistronic construct component specific (intergenic region or PCG) ( Figure 6). showed that PCGs' expression patterns occupying and a unique eigen space, while dicistronic Pearson correlation coefficients were generally higher between the expression of dicistronic 2 0 4 tRNAs and the expression of the intergenic region than between the expression of PCGs and the 2 0 5 expression of the intergenic regions on both tissues (Supplemental Table S5). These correlations were only significant (Pearson correlation, p-value < 0.05) between dicistronic tRNAs and 2 0 7 intergenic regions in leaves (Supplemental Table S5). In this study, using an RNA-seq approach, we found that 15.3% (19/124) of all expressed tRNAs 2 1 0 in grapevine leaf and berry samples were putatively expressed in a dicistronic manner, with 2 1 1 neighbouring protein coding genes. We developed DiRT, a customised, computational pipeline 2 1 2 to specifically detect co-transcribed tRNA and mRNA candidates using stringent criteria. Using DiRT we were able to identify dicistronic transcripts in two different grapevine tissues (i.e. leaf  suggesting conservation amongst plant tRNAs that form dicistronic transcripts. Furthermore, the  Among these tRNA coding for Gly GCC and Met CAT were able to mobilise mRNA transcripts to 2 2 3 different tissues as part of a fusion construct and translate into functional proteins in grafted A. mRNA transcripts to distantly located tissues. A recent study also revealed that mobile RNA determine if the dicistronic tRNAs identified in this study also confer mRNA mobility and to 2 3 3 assess the role of cytosine methylation in mRNA transport in grapevine.

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Interestingly, for four of the 19 dicistronic candidates we also observed sequence conservation such syntenic clusters are conserved through evolution and suggests that these transcripts may have an important functional role.

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Of the 19 dicistronic tRNA genes identified in the Vitis vinifera genome, 18 were located fewer 2 4 0 than 1000 base pairs from the co-transcribed protein coding gene (median distance 133 bp). Our  genomic proximity rule has been found to extend to the dicistronic tRNA-snoRNA genes that Previous studies have indicated that a large proportion of mobile transcripts are also highly these transcripts through the phloem may contribute to their mobility. A significant proportion 2 5 2 (11.4%) of these transcripts was subsequently shown to be dicistronically associated with tRNA formed dicistronic transcripts in grapevine, we did not observe higher abundance of these 2 5 5 transcripts in either tissue analysed. Thus, in our study, the expression level of the tRNA and 2 5 6 mRNA was not a good indicator of the formation of dicistronic transcripts. termination signal suggesting the transcriptional read-through mechanism for these transcripts is are involved in transcribing tRNA-mRNA dicistronic transcripts. The expression patterns of all genomic features studied (i.e. tRNAs, PCGs, and intergenic regions (considered a proxy for dicistronic tRNA-mRNA transcripts) were found to be organ 2 7 8 specific and sensitive to regional environmental differences. The effect of organ and 2 7 9 environment on PCG and tRNA gene expression has been extensively studied before in show that the expression patterns of dicistronic transcript-forming tRNA genes mimic those of 2 8 6 all expressed tRNA (Supplemental Fig S5). We also found that the expression of dicistronic correlation was only statistically significant in leaves, the lack of statistical significance in berry 2 9 0 samples could be due to the low number of dicistronic transcripts identified in berries compared 2 9 1 to leaves (9 vs 16 respectively). Taken collectively, our results suggest that environmentally 2 9 2 1 5 induced dicistronic tRNA-mRNA expression is, at least partially, directed by the mechanisms 2 9 3 regulating tRNA expression.

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Prior to this work, dicistronic transcripts had been found in a handful of plant species grown 2 9 5 under laboratory conditions. Comparative analysis of dicistronic transcripts in grapevine and A. the plant kingdom is needed to begin to understand the biological roles that these types of 2 9 9 transcripts may play in plants. We provide here the first evidence for discistronic transcription in 3 0 0 a commercially important crop species grown in field conditions, and highlight that differential  genome reference IGGP_12X obtained from EnsemblPlants 36 was performed using Hisat2 indexed BAM files were then directly input into the R environment in order to identify the 3 4 0 dicistronic tRNA-mRNA transcripts.  e1003118. by seed-and-vote. Nucleic Acids Res 41: e108. non-homologous microRNAs target transcripts encoding functionally related proteins.

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Genome Biol 10: R136. Identification of grapevine microRNAs and their targets using high-throughput  RNAs reveals the presence of dicistronic snoRNA-miRNA genes in Arabidopsis thaliana. Information Services, Government of South Australia.

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Schramm L, Hernandez N. 2002. Recruitment of RNA polymerase III to its target promoters.