OSU Biochemistry and Molecular Biology

Ramanjulu Sunkar 's Laboratory Research


    Welcome        Research       Lab Members           CV          Dr. Ramanjulu Sunkar  

Role of MicroRNA-directed gene regulation in plant stress tolerance

Proteins are the building blocks of all living cells. The type of cell, its function and the timing of its death are determined by which proteins are produced in the cell, and at what quantities and time they are produced. For example, specific proteins will determine when a plant will flower or initiate a new leaf. However, proteins are the end product of a complex process, which begins with the genetic code present in DNA, which will be copied into mRNA and this holds the instructions on how to build a specific protein. This process is regulated at multiple levels, including transcription, mRNA stability and translation as well as protein stability and protein modifications also contribute to the correct gene expression programs. MicroRNAs regulate when, where and how much of a protein needs to be made by degrading certain number of the number of mRNA molecules while leaving the rest for protein production. In other words microRNAs serve as quantity controllers of protein production. Thus, understanding microRNA-guided gene regulation not only broadens our basic understanding of posttranscriptional gene regulation but also has potential for biotechnological approaches in crop plants such as improving stress tolerance.

Only recently discovered about 10 years ago, microRNAs and small-interfering RNAs (siRNAs) have recently emerged as key regulators of gene expression in eukaryotes. miRNAs are genome-encoded 21 nucleotide noncoding RNAs processed from longer sequences that adopt hairpin-like structures by the Dicer family (Dicer-Like 1) of enzymes. Once processed these miRNAs are loaded into the RNA-induced silencing complex (RISC) required to regulate the expression of their messenger RNA (mRNA) targets via degradation and/or translational suppression of target mRNA. siRNAs are processed from longer double-stranded RNAs by other members of the Dicer family (DCL2, DCL3 and DCL4) of enzymes. Upon processing, they are incorporated into RITS (RNA induced transcriptional silencing complex). These small RNAs serve as the specificity components of RITS, as they guide these complexes to homologous DNA sequences for transcriptional gene silencing. The underlying theme of small RNA-guided gene regulation is that it relies on specific interactions between small RNAs and mRNAs leading to post-transcriptional gene silencing or between small RNAs and DNA leading to transcriptional gene silencing.

Small RNAs, particularly miRNAs have been found to play a central role in plant development. Recently small RNAs have also been found to play important roles in plant stress responses. Research in my laboratory is directed to identify and understand the roles of endogenous small RNAs (microRNAs and small-interfering RNAs) in plant abiotic stress responses. We use high-throughput sequencing of small RNA libraries to identify miRNAs that are altered in response to stress. Similarly, to identify miRNA targets we use degradome analysis, i.e., sequencing of cleaved mRNA targets. Identification of small RNA-guided gene regulation that is important for stress tolerance will provide new tools for improving plant stress tolerance.

 Epigenetic changes and plant stress tolerance

Epigenetics is defined as changes in gene expression that are not associated with changes in DNA sequence. It is mainly the result of methylation of DNA and chemical changes in DNA-associated chromosomal proteins such as histones. Epigenetic modifications (DNA methylation and histone modifications) are known to regulate gene expression by bringing changes in the chromatin state.

 Proteins are the end product of a complex process of gene expression, which begins with the genetic code present in DNA that is copied into mRNA (transcription) that holds the instructions on how to build a specific protein (translation). The transcription of a gene depends not only on the DNA sequence and availability of sequence-specific regulatory factors (transcription factors) but also on the presentation of genes within the complex architecture of the chromosome. Eukaryotic DNA is tightly associated with the histone proteins, and this DNA and histone protein complex together constitutes “chromatin”. In order to initiate transcription at specific gene(s), the chromatin needs to be loosened so that transcription factors can bind and recruit RNA polymerase that transcribes the gene. Thus, the chromatin state (open or closed) at specific gene(s) holds the key to whether a gene will be “ON” (opened configuration of DNA that allow transcription factors and RNA polymerase to initiate transcription) or “OFF” (closed configuration of DNA that is not accessible for transcription factors and RNA polymerase). DNA methylation and histone modifications, known as “epigenetic modifications”, regulate gene expression by bringing changes in the chromatin state. For instance, methylation at position 5 of cytosines is a major epigenetic modification of genomic DNA, which is frequently involved in the silencing of genes. Similarly, histone modifications or the so-called "histone code" such as methylation/acetylation at specific positions on histones has the code for gene expression or silencing. These epigenetic modifications have an essential role in regulating genes, dictating when and where they should be expressed or silenced, thus, epigenetics has emerged as a new frontier in molecular biology.

 It has been evident for few years that the altered plant epigenome is critical for adapting to stress. Recent research in my laboratory is directed to identify specific epigenetic changes that contribute to gene expression during stress. We will use genome-wide analysis of cytosine methylation and chromatin immunoprecipitation (ChIP) to identify such epigenetic changes. This knowledge will provide a new layer of gene regulation that is critical for plant survival under stressful conditions.

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