激光植物miRNA講解

1、UV-B Respo nsive MicroRNA Genes in Arabidopsisthalia naXuefeng Zhou ? , Guandong Wang? and Weixiong Zhang 1, ? , ?Departme nt of Computer Scie nee and Engin eeri ng1Departme nt of Gen eticsWashi ngton Uni versity in Sai nt Louis Sai nt Louis, MO 63130-4899, USAAbstractMicroRNAs are small, non-cod in

2、g RNAs that play critical roles in posttran scripti onal gene regulati on. In pla nts, mature microRNAs pair with compleme ntary sites on mRNAs and subse-que ntly lead to cleavage and degradatio n of the mRNAs. Many microRNAs target mRNAs that en code tran scripti on factors, therefore, they regulat

3、e the expression of many down-stream genes. In this study, we carry out a survey of Arabidop-sis microRNA genes in resp onse to UV-B radia-ti on, an importa nt adverse abiotic stress. We de-velop a no vel computati onal approach to ide ntify microRNA genes induced by UV-B radiation and characterize

4、their functions in regulating gene expressi on. We report that in A. thaliana 21mi-croRNA genes in 11microRNA families are up-regulated un der UVB stress con diti on. We also discuss putative tran scripti onal dow n- regulati on pathways triggered by the in duct ion of these mi-croRNA gen es. Moreov

5、er, our approach can be directly applied to miRNAs responding to other abiotic and biotic stresses and exte nded to miR-NAs in other pla nts and metazoa ns.1ln troductio nMicroRNAs are approximately 22-nu cleotide long, non-codi ng RNAs that play critical roles in regulating gene expression at the p

6、ost-transcriptional level (Bartel,2004; He and Han-non, 2004. The discovery of miRNAs (mi-croRNAs has broade ned our perspectives on the mecha ni sms of repressi on of gene expressi on, which is an importa nt regulatory mecha nism me-diati ng many biological processes such as de-velopme nt, cell pro

7、liferation and differentiation. In plants, mature miRNAs base-pair with complementary sites on target mRNAs and subse-quently direct the mRNAs to be cleaved or de-graded. Pla nt miRNAs regulate many genes that are invo Ived in developme ntal con trol, for example, aux in sig nali ng (Bonn etet al.,

8、2004; Jon es-Rhoades and Bartel, 2004, organ polar-ity (Eshedet al., 2001; Kidner and Martienssen, 2004; McConnell et al., 2001, developme nt tran-sitio ns (Aukerma nand Sakai, 2003; Che n, 2004, leaf growth (Palatniket al., 2003 and RNA metabolism (Vaucheretet al., 2004; Xie et al., 2003. Several r

9、ece nt studies showed importa nt functions of miRNAs in resp onse to adverse abiotic stresses (Bariet al., 2006; Jones-Rhoades and Bartel, 2004; Lu et al., 2005; Sunkar and Zhu, 2004. In Arabidopsis, miR399was ide nti?edo be highly expressed un der phosphate starva?These authors con tributed equally

10、 to this research. ?Corresp onding author:zha ,pho ne:ti on (Bariet al., 2006; Chiou et al., 2006; Fu-(314935-8788,fax:(314935-7302.jii et al., 2005 and miR395wasde nti?edto be1in duced un der sulfate starvati on (Jon es-Rhoadesa nd Bartel, 2004. Furthermore, qua ntitative experime nt

11、al an alysis proved that miR393was stron gly in duced un der cold stress (Sun kara nd Zhu, 2004. In Populus, moreover, some miR-NAs can be in duced by mecha ni cal stress and may fun ctio n in critical defe nse systems for structural and mecha ni cal ?tn ess(Luet al., 2005.Many targets genes of miRN

12、As en code tran-scriptio n factors as well (Bartel,2004, each of which further regulates a set of downstream genes. Thus the activation of miRNA genes un-der abiotic stresses will lead to the repressi on of many dow nstream prote incod ing genes and affect physiological resp on ses. Among various en

13、 vir onmen tal factors, light plays a particularly importa nt role. Sun light is not only the en ergy source for pla nt photos yn thesis, but also regulates several pla nt developme ntal processes and some physiological processes, such as photos yn thesis, seas onal and diur nal time sensing (Baroli

14、et al., 2004; Chattopadhyay et al., 1998; Chory et al., 1996; Jiao et al., 2005. Similar to adverse en-vironmental factors, such as drought and salinity, light can also have stress effect on pla nts. It in ter-acts with en doge nous developme ntal programs, hence affect pla nt growth and developme n

15、t. I n order to acclimate un der such con diti ons, spe- ci?cphotoreceptor systems have bee n developed and evolved to mon itor cha nges of light com-positi on (Dun aevaa nd Adamska, 2001; Harvaux and Kloppstech, 2001; Shao et al., 2006. With complex photoreceptors, pla nt can register UV-B radiati

16、on and tran sduce the in formati on to nu-cleus, hence affect gene expressi on (Chattopad-hyay et al., 1998; Jiao et al., 2005; Kimura et al., 2003b. Chan ges in gene expressi on in resp onse to UV-B radiati on in clude reducti on in expressi on and syn thesis of key photos yn thetic prote ins as we

17、ll as perturbati on of expressi ons of the genes invo Ived in defe nse mecha ni sms (Chattopadhyayet al., 1998; Jiao et al., 2005; Kimura et al., 2003b.Regulati on of gene expressi on plays an impor-ta nt role in a variety of biological processes, such as developme nt and resp on ses to en vir onmen

18、 tal stimuli. In pla nts, transcriptional regulation is2mediated by a large nu mber of tran scripti on fac-tors (TFsco ntrolli ng the expressi on of tens or hundreds of target genes in various, sometimes intertwined, signal transduction cascades (Ve n-ter and Botha, 2004; Wellmer and Riechma nn, 200

19、5. Tran scriptio n factor binding sites (TF-BSs are the functional short DNA sequences (cis -elements that determine the timing and lo-cation of transcriptional activities. Many com-putational methods have bee n developed to re-veal relati on ships betwee n gene expressi on pat-ter ns and TFBSs in t

20、he proximal upstream regu-latory regions of the genes of interest. In yeast, motifs with known functions have bee n related to tran scripti onal pathways by statistical an alysis of the occurre nee of known motifs in the promoters of coregulated genes (Bussemakeret al., 2001. However, the prese nce

21、of in dividual motifs is only margi nally indicative of a gene sexpression ptetrn. Extended strategies pursue to optimally pre-dict gene expressi on patter ns with promoter cis -eleme nts and their comb in ati ons. With a system-atic strategy, the expressi on of a large proporti on of genes in S. ce

22、revisiae was accurately predicted based on promoter seque nces (Beera nd Tavazoie, 2004. Although distal regulatory eleme nts other tha n those in proximal upstream promoter regi ons can modulate gene expressi on, a rece nt study erpihasized that the seque nces in the 5 upstreamregi ons of genes wer

23、e of primary importa nce in Arabidopsis gene regulati on (Leeet al., 2006. Speci?cally,promoter seque nces were suf?cie ntto recapture the mRNA expressi on levels for 80%of the TFs studied. This study con? rmedthe i-porta nt role of promoter regi ons in Arabidopsis gene expressi on.In light of this

24、tran scriptome-based perspec-tive and by tak ing adva ntage of the vast available data of geno me-scale microarray expressi on pro-eof prote in-cod ing gen es, we develop an inno-vative computati onal approach to explore the ex-pressi on activity of miRNA genes un der certa in con diti ons. We focus

25、 on ide ntify ing and anno-tat ing miRNAs in A. thalia na which are resp on-sive to UV-B radiati on, and further con sider the regulatory pathways that are probably affected by the putative UV-B in ducible miRNA gen es. Ourapproach is based on the followi ng two observa-ti ons. First, pla nt miRNAs

26、gen erally direct en-donu cleolytic cleavage of target mRNAs (Llaveet al., 2002; Schwab et al., 2005, hence en able rapid cleara nce of target mRNAs whe n they are expressed (Axtella nd Bartel, 2005; Bartel, 2004. Un der a particular con diti on, if an miRNA is up-regulated, its targetsare most like

27、ly to be co-here ntly dow n-regulated. Second, miRNA genes are tran scribed by RNA polymerase II (Houbaviyet al., 2005; Lee et al., 2004; Xie et al., 2005; Zhou et al., 2007. Hence the 5 proximatensoof miRNA genes are the most importantregulatory regi ons, and sig ni ?ca ntci&eleme nts in these regi

28、 ons are importa nt in determ ining the spatial and temporal expressi on patter ns of the miRNA gen es. Therefore, miRNA and protein-coding genes carrying the same or similar cis -elements in their promoters are very likely to be co-regulated under the same condition and conse-quently very likely to

29、 be co-expressed.Although we focus on Arabidopsis UV-B re-sp onding miRNA genes in this study, our ap-proach can be directly applied to pla nt miRNA genes fun cti oning un der other abiotic or biotic stress con diti ons.Table 1:Putative UV-B resp on sive miRNAs. (a:sta ndard deviati ons of p valuesg

30、ene idmiR159/319miR160miR165/166miR167miR169miR170/171miR172miR393miR39 8miR401#targets 12352735632cos ine similarity0.270.630.460.640.660.610.660.280.840.73p value 7.11E-031.42E-021.17E-026.10E-023.60E-051.69E-024.25E-044.33E- 022.44E-042.96E-02stdv 4.30E-051.94E-041.11E-043.47E-047.00E-061.61E-041

31、.70E-052.67E-046.00E-052.71E-0422.1Results and discussi onsUV-B respo nsive miRNAsOne of the bases of our method for ?ndin gstresEesp on sive miRNA genes is that protei n-codi ng genes targeted by the same miRNA are likely to have cohere ntly dow n- regulated expressi on pat-terns. We con sider an m

32、iRNA to be putatively stress-i nducible if the expressi ons of its target genes are cohere ntly repressed and the cohere nee is statistically signi?cantabove athreshold. In this study, we only considered bona ?detarget genes reported in the literature. For each miRNA, pairwise cosine similarities of

33、 the expressi ons of its target genes were computed. We measured the cohere nee of the expressi ons of its target genes by the average pairwise similarity. The statistical sig ni ?ca nceof the cohere nee was assessed with a3p value from a Monte Carlo simulation. Brie?y,for each miRNA with n target g

34、enes, we ?rstcal-culated the average pairwise cosine similarity of the expressions of the target gen es. We the n ran-domly sampled n genes from the whole set of genes that were pro?led,a nd calculated their average pairwise cos ine similarity. We repeated the sampli ng a large nu mber of times, for

35、 in sta nee one millio n times in our study, and took as an em-pirical p value the freque ncy of observ ing a sim-ilarity value larger tha n that of the target genes. For each miRNA, we repeated this simulation 100times, and calculated the average p value and the sta ndard deviati on.Table Ishows pu

36、tative UV-B resp on sive miR-NAs. For each of these miRNAs, its target genes are cohere ntly dow n-regulated, and the cohere nee of their expressi on patter ns is statistically sig ni ?-ca nt. Except miR167whose p value is less tha n 0.07, all can didates have p values smaller tha n 0.05.For miR158,

37、 miR162, miR163, miR168, miR395, miR402, miR403, miR404, miR405a nd miR406, we only found one bona ?detarget gene in the microarray data set, and could not test their cohere nee, thus excluded them from our study.2.2UV-B respo nsive miRNA genesWe applied our computational approach, dis-cussed in Sec

38、tion 3.2, to the microarray gene expressi on data un der UV-B radiati on treat-me nt from the Arabidopsis AtGe nExpressproject (/i nfo/expressio n/ATGe nExpress.jsp. We predicted 21miRNA genes in 11miRNA families to be up-regulated under UV-B radiation. Table 2lists these UV-B in

39、ducible miRNA gen es. A pu-tative UV-B respo nsive miRNA gene must satisfy two criteria:First, the set of protein-coding genes with the same array of motifs in their proximal promoter regions is enriched with UV-B up-regulated genes. Secon d, its in ferred expressi on (discussedbelow should be an ti

40、-correlated with the expressi ons of its target gen es.For each miRNA gene, we an alyzed whether the combirtio n of sig ni?ca ntmotifs in its pro-moter was statistically releva nt to the UV-B stress. First, we exam ined all protei n- cod ing genes in the whole set of gene pro?led in the microarray e

41、xperime nts, and found those genes that contain the same or very similar motifs in their proxi-mal promoter regi ons. We the n tested whether these prote in-cod ing genes were en riched with up- regulated genes (seeSecti ons 3.2a nd 3.5. We further imposed on miRNA genes a criterion of an ti-correla

42、ti on betwee n the in ferred ex-pressi on of an miRNA gene and the expressi ons of its mRNA targets, in order to ?lterout possible false predicti ons. Since we did not rely on any direct information of miRNA expression, we used the inferred expression of an miRNA gene and the expressions of its targ

43、ets to com-pute their anti- correlatio n (seeSect ion 3.4. I n our study, we chose the ?vebest protecod ing genes whose 5 proximal promoters contain-rarys of cis -elements that most resemble that of the corresp onding miRNA gen es. These ?vege nes are moSkely to be co-regulated with the corre-spondi

44、ng miRNA gene, thus their expression pat-terns are most likely to be similar to the expres-sion pattern of the miRNA gene. In the rest of our discussion, we refer to the average expressi on patter n of the topVfeco-regulated prote in-cod ing genes of an miRNA as its in ferred expressi on pat-tern or

45、 expressi on patter n for short.Before we in ferred expressi on patter ns of miRNA gen es, we applied the in ferri ng proce-dure to 100ra ndomly selected protei n-codi ng genes with known expressi on patter ns, and the n4Table 2:Putative UV-B resp on sive miRNA gen es. (a:p values for assess ing the

46、 en richme nt of UVB up-regulated genes in the set of codi ng genes that contain the same arrays of motifs as the miRNA gen es. (bp values for asess ing the sig ni ?ca nceof the cosine similaries. (c:Standard deviations of the p values (b .gene id miR156e miR156f miR156h miR157c miR159a miR159b miR1

47、60c miR165a miR166c miR166f miR167d miR169d miR169j miR170miR171a miR172c miR172e miR393a miR398a miR401miR395c miR395ep value 2.48E-059.39E-021.67E-068.93E-028.51E-029.94E-043.48E-042.11E- 114.55E-022.50E-072.52E-061.79E-025.36E-101.27E-024.66E-021.16E-028.21E- 051.08E-054.89E-021.02E-128.09E-028.6

48、3E-03cos ine similarity-0.42-0.42-0.41-0.32-0.41-0.48-0.53-0.52-0.47-0.47-0.72-0.41-0.41-0.69-0.75-0.75- 0.77-0.60-0.78-0.71p value 5.81E-022.69E-025.76E-028.32E-023.09E-021.25E-027.14E-025.24E- 027.50E-029.45E-027.81E-029.10E-029.37E-022.82E-028.32E-031.15E-032.60E- 044.52E-025.94E-028.51E-02stdv 1

49、.76E-041.05E-042.73E-042.76E-041.95E-041.11E-043.51E-042.10E- 041.60E-043.64E-041.77E-043.21E-041.93E-041.83E-049.10E-054.20E-051.80E- 051.66E-042.13E-041.97E-04assessed the similarities betwee n their in ferred and actual expressi on patter ns. For all these 100genes, the cosine similarity values o

50、f their in-ferred and actual expression patter ns are betwee n 0.3a nd 0.89, and the average of these values is 0.51. Figure 1shows the in ferred expressi on pat-tern and the actual expressi on pattern of protein cod ing gene, At1g19770. This ?guregives a pic-torial view of the similarity of the inf

51、erred and original expressi on patter ns. The cos ine simi-larity value of these two expressi on patter ns of At1g19770is 0.76.For each putative UV-B resp on sive miRNA gene, we calculated the average cos ine similar-ity betwee n its in ferred expressi on and the ex-pressi ons of its targets. We ass

52、essed the statistical signi?canceof the similarity with a p value. Similar to the analysis of expressi on cohere nee of target genes in Section 2.1, the p value was also-AT1G19770AT1G2555OAT1G60040AT4G 29050 AT5G2O23OAT5G395B0Expressi on pattern of AT1G19770 and its top 5 correlated gen esFigure 1:T

53、he expressi ons of At1g19770in root and shoot, respectively, the expressi ons of the ?veprote in-cod ing genes that are most correlated to it, and their mean expressi on. obtained by a Monte Carlo simulation. We took as an empirical p value the frequency to observe a cosine similarity value smaller

54、than that in the real data. For each miRNA, the simulation was also repeated 100times to obtain an average p value and a standard deviatio n.Forty miRNA genes satisfy the ?rstcriteri on. However, as show n in Table 2, i nferred expres-si ons of 21miRNA genes are an ti-correlated to the expressi ons

55、of their target genes (cos in esim-ilarity less tha n 0, and the an ti-correlati ons r?ectedby the average cos ine similarities of in-ferred expressi ons and expressi ons of target genes are statistically signi?cant.These 21genes are our predicted UVB responsive miRNA genes. In all putative UV-B res

56、ponsive miRNA fami-lies shown in Table 1, at least one member gene from each family was predicted to be up-regulated un der UV-B radiati on. However, none of the members in other miRNA families was predicted to be UV-B responsive. Three miRNA genes, miR168a , miR395c , and miR395e , might also be5UV

57、-B resp on sive. The arrays of motifs in their proximal promoter regi ons are statistically sig ni f-ica ntly releva nt to UV-B stress, show n by small p values obta ined from an accumulative hyper-geometric test. Prote in-cod ing genes shari ng the same array of motifs with them have en riched GO (

58、Ge neOn tology terms that are related to stress resp onse (seediscussi on in Secti on 2.3. How-ever, since these miRNAs have fewer tha n two experime ntal validated target gen es, the coher-e nee of their target gene expressi ons and the an ti-correlatio ns betwee n their expressi ons and ex-pressi

59、ons of their targets could not be an alyzed. Hence these genes will not be discussed further.Table 3:Stress-related GO terms are en riched in the anno tati ons of protei n-codi ng genes that contain all the motifs prese nt in corresp onding putative UV-B resp on sive miRNA gen es.2.2E-52.3E-52.4E-53.1E-53.9E-51.4E-56.8E-56.5E-104.2E-87.8E-71.2E-61.3E- 67.9E-62.5E-55.0E-51.6E-47.0E-64.8E-57.3E-59.4E-53.2E-83.8E-89.5E-71.2E-61.4E- 63.3