Recent findings on MIR163 led to the hypothesis that plant miRNAs may be de novo created by inverted duplication of its targets and subsequent evolutionary drift to lead to functional miRNAs in their present form (Allen et al., 2004). My results given above imply that duplication of miRNAs also involves at least part of the promoters. Thus far it has not been shown whether the initial formation of miRNAs by a mechanism involving inverted duplication also involves duplication of parts of the promoters and to what extent promoter elements and regions are retained between the target gene promoter and the
respective
Figure 2.13
Sequence similarities within the promoter regions of MIR163 and its target genes.
The light blue horizontal bars represent upstream sequences. The dark blue bars at the right end of light blue bars indicates the 50-‐UTR of target genes or 50 ends of pri-‐miR163 which is not part of the microRNA precursor. The red horizontal bars represent the identified TATA box. Colored bars represent conserved regions detected. The peaks represent the number of hits per nucleotide normalized by the number of total hits per sequence, which were calculated 50 times by MotifSampler. A peak is plotted in red when the height of the peak is >50%.
miRNA promoter (Allen et al., 2004). In order to answer these questions I aligned the pri-‐miRNA163 plus 1000bp of promoter sequence to the respective UTRs and promoter sequences from the miR163 target genes, At1g66690, At1g66700, At1g66720 and At3g44860 (Figure 2.13). Numerous conserved regions as well as conserved short sequence motifs are present between the 5’ upstream sequences of MIR163 and its target genes. Beside the conserved transcribed region additional conserved regions located within the promoter have been detected. The region depicted in red (Figure 2.13) harbours the TATA box consensus sequence which can be found in all target genes as well as for MIR163. Our findings on the localisation of the TATA box are consistent with recent results published by (Xie et al., 2005). Many additional promoter regions which are shared between the target genes and MIR163 have been detected (Figure 2.13). These regions have supposedly been retained since the initial inverted duplication event during the formation of MIR163. Thus they most likely represent functionally important regions which might be important for correct transcriptional regulation of MIR163 as well as the target genes. However, further experiments are needed to elucidate the functionality of these motifs.
In contrast to MIR163, there weren’t any significant motifs for MIR161 and its target genes, although the stem loop of MIR161 has also been shown to have a similarity to its target genes (Allen et al., 2004). The same promoter analysis I was performed for non-‐conserved miRNA multigene families MIR157, MIR158, MIR165 and their corresponding targets as listed in ASRP Database (Gustafson et al., 2005). The results are included in our website. No significant sequence similarities were detected by DiAlign for MIR157, MIR158 and MIR165 and their targets, respectively. I then compared the similarity of the promoter regions of non-‐conserved miRNA families with those of all miRNA families, non-‐conserved miRNA families and their targets. I used the similarity of the promoter regions of all miRNA families and random promoter regions as the control group. The similarity of the promoter regions of non-‐conserved families is considerably higher than other groups (Figure 2.14), which is supportive of our hypothesis that the duplication of non-‐conserved miRNAs had included the promoter regions of miRNAs.
Figure 2.14
Compare the alignability of the promoter regions of non-‐conserved miRNA families with those of all miRNA families, non-‐conserved miRNA families and their targets.
The role of miRNAs as a part of gene regulatory machinery has been demonstrated by recent intensive research (Bartel, 2004; Baulcombe, 2005). However, the transcriptional regulation of miRNA genes themselves has not been the subject of extensive research. My analysis, which detected similarities within the precursor as well as the conserved regions within the promoter sequences, provides additional evidence to support the inverted duplication model for plant microRNA evolution. In addition, the detected conserved miRNA upstream motifs represent prime candidates to study the regulation of
Arabidopsis MIR157, 158, 163, 165, 405 and 447. These finding suggests that the initial inverted
duplication event, which had created miRNA from its targets, has included the promoter region of target genes. For expanded miRNA families regulatory diversification through modulation of cis regulatory elements might be a mechanism leading to fixation of sequence redundant miRNA genes. In analogy to duplicated protein-‐coding genes (Hurles, 2004) promoter elements seem to be shared within paralogous MIR genes. This permits to sketch a powerful in silico approach which might be considered for the experimental and in silico analysis of microRNA promoters.
This chapter mainly focus on miRNA genes. The topics include finding miRNA genes on a genome and promoter analysis of miRNA genes and their targets. In the next chapter, I will present another kind of small RNAs, viral derived small RNAs (vsRNAs).
Reference
Allen, E., Xie, Z., Gustafson, A.M., Sung, G.H., Spatafora, J.W., and Carrington, J.C. (2004). Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat Genet 36, 1282-‐1290.
Axtell, M.J., Snyder, J.A., and Bartel, D.P. (2007). Common functions for diverse small RNAs of land plants. Plant Cell 19, 1750-‐1769.
Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-‐297. Baulcombe, D. (2005). RNA silencing. Trends Biochem Sci 30, 290-‐293.
Bonnet, E., Wuyts, J., Rouze, P., and Van de Peer, Y. (2004). Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proc Natl Acad Sci U S A
101, 11511-‐11516.
Borchert, G.M., Lanier, W., and Davidson, B.L. (2006). RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 13, 1097-‐1101.
Chaudhury, A. (2005). Plant genetics: hothead healer and extragenomic information. Nature 437, E1; discussion E2.
Griffiths-‐Jones, S. (2004). The microRNA Registry. Nucleic Acids Res 32, D109-‐111.
Gustafson, A.M., Allen, E., Givan, S., Smith, D., Carrington, J.C., and Kasschau, K.D. (2005). ASRP: the Arabidopsis Small RNA Project Database. Nucleic Acids Res 33, D637-‐640.
Haberer, G., Hindemitt, T., Meyers, B.C., and Mayer, K.F. (2004). Transcriptional similarities, dissimilarities, and conservation of cis-‐elements in duplicated genes of Arabidopsis. Plant Physiol 136, 3009-‐3022.
Hindemitt, T., and Mayer, K.F. (2005). CREDO: a web-‐based tool for computational detection of conserved sequence motifs in noncoding sequences. Bioinformatics 21, 4304-‐4306.
Hurles, M. (2004). Gene duplication: the genomic trade in spare parts. PLoS Biol 2, E206.
Jones-‐Rhoades, M.W., and Bartel, D.P. (2004). Computational identification of plant microRNAs and their targets, including a stress-‐induced miRNA. Mol Cell 14, 787-‐799.
Kurihara, Y., and Watanabe, Y. (2004). Arabidopsis micro-‐RNA biogenesis through Dicer-‐like 1 protein functions. Proc Natl Acad Sci U S A 101, 12753-‐12758.
Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H., and Kim, V.N. (2004). MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23, 4051-‐4060.
Li, W.X., Oono, Y., Zhu, J., He, X.J., Wu, J.M., Iida, K., Lu, X.Y., Cui, X., Jin, H., and Zhu, J.K. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. Plant Cell 20, 2238-‐2251.
Llave, C., Xie, Z., Kasschau, K.D., and Carrington, J.C. (2002). Cleavage of Scarecrow-‐like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053-‐2056.
Lolle, S.J., Victor, J.L., Young, J.M., and Pruitt, R.E. (2005). Genome-‐wide non-‐mendelian inheritance of extra-‐genomic information in Arabidopsis. Nature 434, 505-‐509.