[EN] miRNAs are fundamental endogenous regulators of gene expression in higher organisms. miRNAs modulate multiple biological processes in plants. Consequently, miRNA accumulation is strictly controlled through miRNA precursor accumulation and processing. Members of the miRNA319 family are ancient ribo-regulators that are essential for plant development and stress responses and exhibit an unusual biogenesis that is characterized by multiple processing of their precursors. The significance of the high conservation of these non-canonical biogenesis pathways remains unknown. Here, we analyze data obtained by massive sRNA sequencing and 5 ' - RACE to explore the accumulation and infer the processing of members of the miR319 family in melon plants exposed to adverse environmental conditions. Sequence data showed that miR319c was down regulated in response to low temperature. However, the level of its precursor was increased by cold, indicating that miR319c accumulation is not related to the stem loop levels. Furthermore, we found that a decrease in miR319c was inversely correlated with the stable accumulation of an alternative miRNA (#miR319c) derived from multiple processing of the miR319c precursor. Interestingly, the alternative accumulation of miR319c and #miR319c was associated with an additional and non-canonical partial cleavage of the miR319c precursor during its loop-to-base-processing. Analysis of the transcriptional activity showed that miR319c negatively regulated the accumulation of HY5 via TCP2 in melon plants exposed to cold, supporting its involvement in the low temperature signaling pathway associated with anthocyanin biosynthesis. Our results provide new insights regarding the versatility of plant miRNA processing and the mechanisms regulating them as well as the hypothetical mechanism for the response to cold-induced stress in melon, which is based on the alternative regulation of miRNA biogenesis. Bustamante-González, AJ.; Marques Romero, MC.; Sanz-Carbonell, A.; Mulet, JM.; Gomez, GG. (2018). Alternative processing of its precursor is related to miR319 decreasing in melon plants exposed to cold. Scientific Reports. 8:1-13. https://doi.org/10.1038/s41598-018-34012-7 Borges, F. & Martienssen, R. A. The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol 16, 727–741 (2015). Shriram, V., Kumar, V., Devarumath, R. M., Khare, T. S. & Wani, S. H. MicroRNAs As Potential Targets for Abiotic Stress Tolerance in Plants. Front Plant Sci 7, 817 (2016). Xie, M., Zhang, S. & Yu, B. microRNA biogenesis, degradation and activity in plants. Cell Mol Life Sci 72, 87–99 (2015). Bologna, N. G., Schapire, A. L. & Palatnik, J. F. Processing of plant microRNA precursors. Brief Funct Genomics 12, 37–45 (2012). Achkar, N. P., Cambiagno, D. A. & Manavella, P. A. miRNA Biogenesis: A Dynamic Pathway. Trends Plant Sci 21, 1034–1044 (2016). Dong, Z., Han, M. H. & Fedoroff, N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci USA 105, 9970–9975 (2008). Bologna, N. G. et al. Multiple RNA recognition patterns during microRNA biogenesis in plants. Genome Research 23, 1675–1689 (2013). Baranauskė, S. et al. Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins. Nucleic Acids Res 43, 2802–2812 (2015). Zhang, S., Liu, Y. & Yu, B. New insights into pri-miRNA processing and accumulation in plants. WIREs. RNA 6, 533–545 (2015). Ren, G. et al. Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc Natl Acad Sci USA 109, 12817–12821 (2012). Cuperus, J. T., Fahlgren, N. & Carrington, J. C. Evolution and functional diversification of MIRNA genes. Plant Cell 23, 431–442 (2011). Zhang, W. et al. Multiple distinct small RNAs originate from the same microRNA precursors. Genome Biol 11(8), r81 (2010). Addo-Quaye, C. et al. Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome. RNA 15, 2112–2121 (2009). Axtell, M. J., Snyder, J. A. & Bartel, D. P. Common functions for diverse small RNAs of land plants. Plant Cell 19, 1750–1769 (2007). Bologna, N. G., Mateos, J. L., Bresso, E. G. & Palatnik, J. F. A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J 28, 3646–3656 (2009). Li, Y., Li, C., Ding, G. & Jin, Y. Evolution of MIR159/319 microRNA genes and their post-transcriptional regulatory link to siRNA pathways. BMC Evol Biol 11, 122 (2011). Sobkowiak, L., Karlowski, W., Jarmolowski, A. & Szweykowska-Kulinska, Z. Non-Canonical Processing of Arabidopsis pri-miR319a/b/c Generates Additional microRNAs to Target One RAP2.12 mRNA Isoform. Front Plant Sci 3, 46 (2012). Achard, P., Herr, A., Baulcombe, D. C. & Harberd, N. P. Modulation of floral development by a gibberellin-regulated microRNA. Development 131, 3357–3365 (2004). Allen, R. S. et al. Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family. Proc Natl Acad Sci USA 104, 16371–16376 (2007). Jones-Rhoades, M. W. & Bartel, D. P. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14, 787–799 (2004). Palatnik, J. F. et al. Control of leaf morphogenesis by microRNAs. Nature 425, 257–263 (2003). Wang, S. T. et al. MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa). PLoS One 9(3), e91357 (2014). Thiebaut, F. et al. Regulation of miR319 during cold stress in sugarcane. Plant Cell Environ 35, 502–512 (2012). Sunkar, R. & Zhu, J. K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 2001–2019 (2004). Chen, H. et al. A comparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance: Solanum lycopersicum and Solanum habrochaites. BMC Plant Biol 15, 132 (2015). Garcia-Mas, J. et al. The genome of melon (Cucumis melo L.). Proc Natl Acad Sci USA 109, 11872–11877 (2012). Nuñez-Palenius, H. G. et al. Melon fruits: genetic diversity, physiology, and biotechnology features. Crit Rev Biotechnol 28, 13–55 (2008). Gonzalez-Ibeas, D. et al. Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing. BMC Genomics 12, 393 (2011). Herranz, M. C., Navarro, J. A., Sommen, E. & Pallas, V. Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems. BMC Genomics 16, 117 (2015). Sattar, S. et al. Expression of small RNA in Aphis gossypii and its potential role in the resistance interaction with melon. PLoS One 7(11), e48579 (2012). Dai, X. & Zhao, P. X. psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res. 39, W155–9 (2011). Palatnik, J. F. et al. Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319. Dev Cell 13, 115–125 (2007). He, Z., Zhao, X., Kong, F., Zuo, Z. & Liu, X. TCP2 positively regulates HY5/HYH and photomorphogenesis in Arabidopsis. J Exp Bot 67, 775–785 (2016). Lau, O. S. & Deng, X. W. Plant hormone signaling lightens up: integrators of light and hormones. Curr Opin Plant Biol 13, 571–577 (2010). Oyama, T., Shimura, Y. & Okada, K. The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl. Genes Dev 11, 2983–2995 (1997). Ahmed, N. U., Park, J. I., Jung, H. J., Hur, Y. & Nou, I. S. Anthocyanin biosynthesis for cold and freezing stress tolerance and desirable color in Brassica rapa. Funct Integr Genomics 15, 383–394 (2015). Catalá, R., Medina, J. & Salinas, J. Integration of low temperature and light signaling during cold acclimation response in Arabidopsis. Proc Natl Acad Sci USA 108, 16475–16480 (2011). Schulz, E., Tohge, T., Zuther, E., Fernie, A. R. & Hincha, D. K. Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions. Plant Cell Environ 38, 1658–1672 (2015). Perea-Resa, C., Rodríguez-Milla, M. A., Iniesto, E., Rubio, V. & Salinas, J. Prefoldins Negatively Regulate Cold Acclimation in Arabidopsis thaliana by Promoting Nuclear Proteasome-Mediated HY5 Degradation. Mol Plant 10, 791–804 (2017). Solfanelli, C., Poggi, A., Loreti, E., Alpi, A. & Perata, P. Sucrose-Specific Induction of the Anthocyanin Biosynthetic Pathway in Arabidopsis. Plant Physiol 140, 637–646 (2006). Reis, R. S., Eamens, A. L. & Waterhouse, P. M. Missing Pieces in the Puzzle of Plant MicroRNAs. Trends Plant Sci 20, 721–728 (2015). Kumar, R. Role of microRNAs in biotic and abiotic stress responses in crop plants. Appl Biochem Biotech 174, 93–115 (2014). Ma, C., Burd, S. & Lers, A. miR408 is involved in abiotic stress responses in Arabidopsis. Plant J 84, 169–187 (2015). Song, L., Axtell, M. J. & Fedoroff, N. V. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol 20, 37–41 (2010). Bracken, C. P. et al. Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage. Nucleic Acids Res 39, 5658–5668 (2011). Gurtan, A. M., Lu, V., Bhutkar, A. & Sharp, P. A. In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs. RNA 18, 1116–1122 (2012). Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet Journal 17, 10–12 (2011). Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq-2. Genome Biol 15, 550 (2014). Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11, 3–r25 (2010). Griffiths-Jones, S. miRBase: microRNA sequences and annotation. Current protocols in bioinformatics 12, 9 (2010). Li, H. et al. 1000 Genome Project Data Processing Subgroup The sequence alignment/map format & SAMtools. Bioinformatics 25, 2078–2079 (2009). Quinlan, A. & Hall, I. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001). Llave, C., Xie, Z., Kasschau, K. D. & Carrington, J. C. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056 (2002). Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, 3–r25 (2009).

Alternative processing of its precursor is related to miR319 decreasing in melon plants exposed to cold

Abstract