Six Emphasis Type="Italic"Medicago truncatula/E

Authors: Aleksander Tworak Anna Urbanowicz Jan Podkowinski Anna KurzynskaKokorniak Natalia Koralewska Marek Figlerowicz

Publish Date: 2016/01/29

Volume: 35, Issue: 5, Pages: 1043-1052

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Abstract

Over the past decade small noncoding RNAs sncRNA have emerged as widespread and important regulatory molecules influencing both the structure and expression of plant genomes One of the key factors involved in sncRNA biogenesis in plants is a group of RNase IIItype nucleases known as Dicerlike DCL proteins Based on functional analysis of DCL proteins identified in Arabidopsis thaliana four types of DCLs were distinguished DCL14 DCL1 mainly produces 21 nt miRNAs The products generated by DCL2 DCL3 and DCL4 belong to various classes of siRNAs that are 22 24 and 21 nt in length respectively M truncatula is a model legume plant closely related to many economically important cultivable species By screening the recent M truncatula genome assembly we were able to identify three new DCL genes in addition to the MtDCL13 genes that had been earlier characterized The newly found genes include MtDCL4 and two MtDCL2 homologs We showed that all six M truncatula DCL genes are expressed in plant cells The first of the identified MtDCL2 paralogs encodes a truncated version of the DCL2 protein while the second undergoes substantial and specific upregulation in the root nodules Additionally we identified an alternative splicing variant of MtDCL1 mRNA similar to the one found in Arabidopsis Our results indicate that DCL genes are differently activated during Medicago symbiosis with nitrogen fixing bacteria and upon pathogen infection In addition we hypothesize that the alternative splicing variant of MtDCL1 mRNA may be involved in tissuespecific regulation of the DCL1 levelSmall noncoding RNAs sncRNAs including microRNAs miRNAs and several types of small interfering RNAs siRNAs are among the most important factors influencing both the structure and expression of eukaryotic genomes SzweykowskaKulińska et al 2003 SncRNAs are involved in the regulation of numerous biological processes such as development stress response and pathogen defense KurzyńskaKokorniak et al 2009 Jackowiak et al 2011 Castel and Martienssen 2013 Martínez de Alba et al 2013 Biogenesis of both miRNAs and siRNAs depends on a group of endonucleolytic enzymes that are members of the RNase III family known as Dicer proteins in animals KurzynskaKokorniak et al 2015 and Dicerlike DCL proteins in plants Bologna and Voinnet 2014 Arabidopsis thaliana a model dicot plant encodes four DCL proteins AtDCL14 Each of these represents a distinct type of DCL found in plants All four types are believed to have diverged early in plant evolution Mukherjee et al 2013 AtDCL1 is mainly responsible for the production of 21 nt miRNAs while AtDCL2 AtDCL3 and AtDCL4 produce different classes of siRNAs Xie et al 2004 However the functions of all DCL enzymes are partially redundant Gasciolli et al 2005 In many plants duplications of the genes encoding the DCL proteins have been observed For example the rice genome encodes two DCL2 and two DCL3 isoforms Kapoor et al 2008 whereas in soybean two isoforms each of DCL1 DCL2 and DCL4 were identified Curtin et al 2012Plant DCL proteins share a common domain architecture with their animal counterparts A typical DCL protein contains an Nterminal helicase domain DExD/Hbox and helicaseC subdomains followed by DUF283 domain of unknown function known also as Dicer dimerization domain PAZ PiwiArgonauteZwille tandem RNase III domains and one or two Cterminal dsRBDs dsRNA binding domains The major catalytic activity of DCL enzymes is provided by the two RNase III domains which cleave dsRNA substrates and release short RNA duplexes with 2 nt 3′ overhangs and phosphorylated 5′ ends In the miRNA biogenesis pathway DCL1 produces duplexes formed by two 21 nt RNA strands miRNA and a complementary passenger strand referred to as miRNA Both PAZ and the helicase domains are known for their role in proper positioning of the sncRNA precursor within the enzyme Macrae et al 2006 Gu et al 2012 while the helicase also allows processive cleavage of longer substrates Cenik et al 2011 Welker et al 2011The expression patterns of different DCL genes vary depending on the tissue or environmental conditions Liu et al 2009 Capitão et al 2011 Two miRNAs miR162 and miR838 were shown to negatively regulate the level of DCL1 expression in Arabidopsis Both are involved in negative feedback loops that control the level of DCL1 in plant cells Conserved miR162 targets the AtDCL1 mRNA within the exon 12–13 junction The biogenesis of nonconserved miR838 located in intron 14 of the AtDCL1 mRNA precursor was proposed to interfere with proper splicing of AtDCL1 transcript Xie et al 2003 Rajagopalan et al 2006 However the latter phenomenon was discovered in a single plant species and the question of whether this is a general mechanism or specific to the selected species has not yet been addressed

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