Diversity Of Endogenous Non-Coding Small RNA From Rice

Organisms have many types of small non-coding RNAs, which involved in degrading mRNA, repressing translation, modifying chromatins, defending against viruses and transposons. The different functions are carried out by different types small RNA. The diversity of small RNA implies that they played various roles in the development of cells. Diversity of small RNA exits in rice according to results analyzed the small RNA population from cDNA libraries. The results are as follows:1. Total RNA was abstracted from leaves and anthers of 9311 (Oryza sativa indica.). 15-35nt small RNAs were isolated and added an adaptor to their 5' and 3' end respectively. Then reverse transcription products were amplified with PCR. PCR products were digested with EcoR I and ligated. 200-800 nt fragments were picked up and transformed into pGEM-T vector. Then cDNA libraries of the small RNA were constructed.2. Single clone was selected from the cDNA libraries. Totally, 169 clones (anther, 114 clones and leave, 55 clones) were sequenced and encoded 162 unique sequences which range in length from 16 to 35 nt. The majority of these RNA are 21-24 nt. showing two peaks at 21-22 nt and 24 nt. Comparison of these small RNA sequences to the genome sequences of rice (indica. and japonica.) identify completely genomic matches for 129 (79.6%) of the small RNA sequences, which include 47 derived from housekeeping non-coding RNAs (10 tRNA, 34 rRNA, 1 snRNA and 2 snoRNA); Two match to sequences from other plant chloroplast DNA. The other 31 (19.1%) contain one or more internal mismatches.3. Various sequences of the small RNA decide the diversity of their targets and exhibit their diverse functions. Their targets are identified with miRU soft, which involve transposon and retrotransposon protein, transcription factor, F-box protein family, Leucine Rich Repeat, Plant protein family, translation initiation factor eIF-5A, embryogenesis transmembrane protein, no apical meristem family protein, DNA-directed RNA polymerase II subunit,polyadenylate binding protein, chromatin structure regulator, ubiquitin-conjugating enzyme, ubiquitin carboxyl- terminal hydrolase, DEAD/DEAH box helicase, potassium channel regulatory factor, Piwi domain, putative disease resistant protein, DNA mismatched repair protein, expressed protein and so on.4. Small RNAs were mapped to the rice chromosomes with Vector NTI 8 soft based on the blast searching of small RNA in 9311 database. The distribution of small RNA correlated with the length of chromosomes, the longest chromosome I has the most loci, while the shortest chromosome X has the least loci. Furthermore, we found that small RNAs arc in or near almost the repeat regions. 20 rasiRNA lie in repeat regions, and the first 19nt of m42 (24 nt) are homogenous with Repeat 231440 in antisense. Others are within a distance of 1000 nt from the repeat regions. Very few are 10000 nt away from the repeat regions; for example, ml 14 is 12647 nt downstream from Repeat233384, the farthest away from a repeat region. Interestingly, we found 13 small RNA are clustered on DNA fragment, which resulted from 5880 nt chloroplast DNA fragments laterally transferred into nucleus. Those small RNA distribute on chromosomes either together or in small clusters and their distribution are different between indica and japanica. Additionally, some small RNA of different lengths overlap and share the same base in 5' end.5. A subset of the small RNA sequences that perfectly match to or have single-mismatch similarity to rice genome sequences and do not match to known 47 non-coding RNA in rice (82 perfect matching, 8 single mismatching) was studied further. Of those 90 small RNA molecular, 44 of them match to single locus, other 46 each exhibit multiple hits with a number of targets; collectively, they hit 535 loci of rice genome. Furthermore, 31 out of 90 small RNAs are similar or identical to the sense strands of matched cDNA. There is only one fragment (m88) match to cDNA in antisense, which represents endogenous siRNA. Otherwise, there include 17 miRNAs, 20 rasiRNAs and 30 tncRNAs.6. We analyzed miRNA precursors up to 600 nt for stem-loop structures using mfold. Out of 90 small RNA, 17 were predicted as miRNA candidates according to their precursor structures. Four of them match to previously validated families of miRNA (miR156, miR167. miR168 and miR169). Five of these miRNA were detected by Northern blot analysis. Moreover, five small RNA fragments (ml, m29, m65, m69 and m240) are similar to Group II miRNA in Arabdopsis. The sizes of miRNA range from 18 to 32 nt and precursors arc 60-300 nt in length. Except the four known miRNA, the other 13 miRNA candidates match to one or multiple genomic loci. Four of these (m2, ml5, m38 and ml50) hit a single locus; the rest nine each match to multiple loci. The flanking sequences of each locus are predicted with hairpin structure, representing a precursor of miRNA. Six miRNA candidates (ml, m2, ml 5,m38. m69 and ml50) have only one precursor; the remaining seven had multiple precursors. And the lengths and structures of precursors are conservative for each miRNA family. Five miRNA candidates (m2. m29, m69, ml50 and ml74) are homogenous to repeat sequences, implying that these miRNA are possibly originated from the repeat sequences. m240 and m243 locate on the two arms of the same hairpin structure, but they are likely differentially processed from the same precursor because they are different in length.To predict potential targets of the miRNA identified in this study, various databases for rice EST, cDNA and Pseudomolecules_CDS were searched. 91 potential targets are predicted for 13 miRNA candidates, and m243 has 35 fully complementary targets expressed almost in all the organisms. The target sites are similar to those of other miRNA in plants, which are located in ORF or UTR of mRNA. Four miRNA (ml4, m29, m65 and m243) are completely complementary to stress induced mRNA, ml4 correlated with cold while the other three relate to drought stress. Out of question, those small RNA play a role in response to environmental stress. Two targets are transcriptional factors, ml5 target is a squamosa promoter-binding protein, ml82 target is a putative RING zinc finger protein (3' UTR). Others, m38 target is a SMC family protein and ml target is a ubiquitin carrier protein UBC7. Interesting, ml50 transcribes in an antisense orientation to repeatl47962, which is a LINE type retrotransposon. Furthermore, 360 nt EST complementary to m29 were amplified by RACE PCR, among which 179 nt are homogenous with four known EST (BI306579, BI305844, BE230013, BE229926) , while 90 nt among of these 179 nt have the stem-loop structure.7. Of 90 small RNA, 20 rasiRNA are identified. Ten of them match to repetitive rDNA, eight rasiRNA match to transposable elements sequences and two rasiRNAs match to simple repeat sequences. Other 30 fragments range from 18 to 24 nt in length, which locate in non-coding regions of rice genome and do not meet the criterion for miRNA, may represent the so-called tncRNA in rice. By analyzing the sequences near the small RNA, we identified new pri-miRNA near m3, m54, m83, m93 and m232. Furthermore, there are two pri-miRNA near m3.8. 14 putative RNase III genes were identified in rice by computer analysis. There are three types of RNase III family in rice: six RNase III proteins of type I ; one RNase III protein of type II; seven RNase III proteins of type III, named Dicer-like protein in plant. We cloned a Dicer-like mRNA from spikelets of 9311 by RACE and step-by-step PCR amplified This OsDCL is 5194 nt long, whose 5' UTR is 1646 nt, ORF is 3351 nt, and 3' UTR is 208 nt and includes a ploy (A) in 3! end. With the absence of 88-200lh bases, 5' UTR has at alternative edition. OsDCL is similarity with a putative RNA helicase in rice. This Dicer-liki gene, including 25 extrons and 24 introns, is encoded by 12108nt, which locates oichromosome 10. The longest ORF encodes 1116 amino acids based on ORF finder al NCBI, which includes a DUF283 domain, a PAZ domain, two RNase III and a double strands RNA binding domain. RT-PCR analysis indicated that the OsDCL expressed at rice roots, stems and spikelets.