Genome-wide Profiling And Evolution Of Small RNAs In Rice

It has been known that most eukaryotes contain a diversified set of small RNA-guided pathways that negatively regulate or control genes,repeated sequences, and viruses at the transcriptional and posttranscriptional levels.Owing to the development of better experimental and computational approaches,an ever increasing number of small RNAs are uncovered in different plant genomes.There are two major classes of small RNAs that are found in the plant kingdom,namely small interfering RNAs(siRNAs),microRNAs(miRNAs),each derived from distinct modes of biogenesis and genomic loci.Small RNA-generating loci,especially those producing predominantly 24-nt siRNAs,were found to be highly correlated with repetitive elements across the genome.It was found that there are many different components, such as RNA-dependent RNA polymerase(RDR),Dicer-like enzyme(DCL), Supressor of Gene Silencing(SGS)and Argonaute(AGO),are involved in diverse small RNA pathway in plant.All forms of silencing by small RNAs require an effector protein of the AGO family.Recent advances in technology permit practical deep sequencing of small RNA populations.Several studies applying high-throughput sequencing methods have helped on both qualitative discovery of new small RNAs or small RNA classes,as well as quantitative profiling of small RNA populations. Although our understanding of the diversity,biogenesis and function of these regulatory RNAs is growing rapidly,their evolutionary process is not well understood. Genome duplication is one of the primary accelerators of evolution.It provide a source of genetic material for mutation,drift,and selection to act upon,making new evolutionary opportunities possible.Ancient genome duplication events have been identified in diverse organisms,such as yeast,vertebrates,Arabidopsis and Poaceae (Gramineae,grass family).Recent investigations found that duplication events played an important role in the main mechanisms involved in the diversification and evolution of several Arabidopsis miRNA families,such as the miR159 and miR395 families.Here,we adopt the approach combining experimental and computational methods, to infer the evolution of two typical small RNA family,TAS3 and miR156 in grass family.Using the small RNA data generated from our high-throughput sequencing of small RNA populations of developing rice grain,we analyzed the distribution and expression divergence of small RNA-generating loci in rice genome.trans-acting siRNAs(tasiRNAs)are a plant-specific class of 21-nt endogenous siRNA.It function in a similar manner to miRNAs,cleave target mRNAs via interaction with a target site with non-perfect complementarity.Each tasiRNA locus (known as a TAS gene)produces a non-coding transcript,a portion of which is converted into dsRNA,this in turn is successively cleaved into mostly 21-nt tasiRNAs phased in a 21-nt register.The production of tasiRNAs requires SGS3/RDR6/DCL4 pathway,miRNA function is required to set the phasing register for tasiRNA production.Over ten TAS genes have been characterized to date,from one locus in rice(TAS3)to four loci in Arabidopsis(TAS1-4).Biological functions have been assigned to a TAS3 family tasiRNA,tasiARF,tasiARF downregulates mRNAs encoding Auxin Response Factors(ARFs),including ARF2,ARF3 and ARF4,and is involved in the proper timing of vegetative shoot development and establishment of leaf polarity.The TAS3 family is distinguished from other TAS loci by the dual miR390 complementary sites flanking the tasiRNA region.TAS3 loci have been reported in rice and other seed plants based on their conserved tasiARFs and dual miR390 complementary sites.Computational procedures have also been successfully used to identify small RNAs based on evolutionary conservation.In this study,56 putative TAS3 genes were identified by database mining and PCR amplification. Phylogenetic analysis indicated that at least three genome/gene duplication events have been involved in the expansion of TAS3 genes and that many TAS3 genes have been lost during evolution in the grass family.Sequence analysis reveals that a high conservation of 21-nt changes in length between tasiARF and the 3' miR390 binding site and the presence of TAS3-like loci in a miR390-targeted protein-coding gene (LRR kinase)in rice and Arabidopsis.We discussed the origin and potential mechanisms for the biogenesis of TAS3 genes in the light of these findings。miRNAs are～21-24 nt long.They derive from hairpin structured miRNA precursors(pre-miRNAs)that are processed by DCL1 from primary miRNAs (pri-miRNAs)transcribed by RNA polymeraseⅡ.miRNAs post-transcriptionally down-regulate gene expression by cleavage or translational repression of target mRNAs.In rice,hundreds of miRNAs have been identified by cloning or computational prediction.It was found that duplication was also one of the main mechanisms involved in the evolution of several miRNA families in Arabidopsis.The miR156 family was one of the first characterized miRNA families in plants,and has 12 members in rice.It is highly conserved in the plant kingdom and has been identified in 45 different plant species,miR156 has been demonstrated to target SPL genes,which are plant specific transcription factors containing an SBP box.Of 12 miR156 family members located on six chromosomes in rice genome,miR156b and miR156c gene(MIR156b/c hereafter)are tandem genes on chromosome 1.A full-length cDNA(AK110797)encodes both miRNAs.Over-expression of miR156b resulted in multiple-tillers and bushy phenotype in maize and flee.MIR156b/c locus was highly conserved among cereals,but not in dicots.Genome duplication events played an important role in the evolution of the miR156 family.We performed sequencing on the miR156b/c loci from 45 diverse Oryza accessions,with 15 accessions of O.rufipogon and 30 domesticated lines of O.sativa(15 indica and 15 japonica cultivars).Genetic diversity investigation at the locus indicated that only～9%of nucleotide diversity observed in wild rice(O.rufigogon)was maintained in the cultivated rice and the neutral model was rejected(P＜0.05)based on Tajima's D and Fu and Li's D~* and F~* tests.These results indicated that the MIR156b/c locus experienced strong natural selection in O.rufipogon,and natural and/or domestication selection in the cultivated rice.To study the distribution and expression divergence of small RNA-generating loci in rice genome,we analyzed the small RNA data generated from our high-throughput sequencing of small RNA populations in developing rice grain.Our results showed that small RNA-generating loci from the repeat associated and intergenic region are most abundant and highly active.Scrolling-window analysis along each chromosome showed that some regions spawn relatively high numbers of small RNA.The significant distribution difference of small RNA populations between duplicated segmental pairs in rice genome was found.It suggested that duplication events played an important role in diversification and evolution of small RNA.