RNAi,miRNA

1. By,
R.Abhilash,
2016670801.

2. Gene Expression
 How does an individual cell specify which of its many thousands of genes to express?
 Important problem for multi-cellular organisms because, as the animal develops, cell types
such as muscle, nerve, and blood cells become different from one another, even-tually
leading to the wide variety of cell types seen in the adult.
 Such differentiation arises because cells make and accumulate different sets of RNA and
protein molecules: that is, they express different genes.

3. Regulation of gene expression
 Includes a wide range of mechanisms that are used by cells to increase or decrease the production of
specific gene products (protein or RNA).
 Any step of gene expression can be modulated, from transcriptional initiation, to RNA processing,
and to the post-translational modification of a protein.
 Often, one gene regulator controls another, and so on, in a gene regulatory network.
 Up-regulation - process that occurs within a cell triggered by a signal (originating internal or
external to the cell), which results in increased expression of one or more genes and as a result the
protein(s) encoded by those genes.
 Down-regulation - process resulting in decreased gene and corresponding protein expression.

5. Regulation Systems
 Inducible systems - An inducible system is off unless there is the presence of some molecule
(called an inducer) that allows for gene expression. The molecule is said to "induce expression".
 Repressible systems - A repressible system is on except in the presence of some molecule (called a
corepressor) that suppresses gene expression. The molecule is said to "repress expression".

6.  Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility
and adaptability of an organism by allowing the cell to express protein when needed.
 Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and
Dissociator (Ds), in the color formation of maize seeds.
 lac operon, discovered by Jacques Monod - some enzymes involved in lactose metabolism are
expressed by E. coli only in the presence of lactose and absence of glucose.
 In multicellular organisms, gene regulation drives cellular differentiation and morphogenesis in
the embryo, leading to the creation of different cell types that possess different gene expression
profiles from the same genome sequence.
 Basis for how evolution actually works at a molecular level, and is central to the science
of evolutionary developmental biology.
Prokaryotes vs Eukaryotes

7. Regulated stages of gene expression
 Chromatin domains
 Transcription
 Post-transcriptional modification
 RNA transport
 Translation
 mRNA degradation

8. Modification of DNA
 Structural
Octameric protein complexes called nucleosomes - responsible for the amount
of supercoiling of DNA - can be temporarily modified by processes such as phosphorylation or
more permanently modified by processes such as methylation.
 Chemical
1. Methylation of DNA- DNA is typically methylated by methyltransferase enzymes on
cytosine nucleotides in a CpG dinucleotide sequence.
Analysis of the pattern of methylation - bisulfite mapping.
2. Histone deacetylation - Histone acetyltransferase enzymes (HATs) such as CREB-
binding protein dissociate the DNA from the histone complex, allowing transcription to
proceed.
E.g: FLC and FLD genes in Arabidopsis regulate flowering.

10. Regulation of expression by nitrate in TCA cycle genes
 Nitrate has been shown to be a signal to regulate gene expression in plants. Four microarray
experiments with wild-type seedlings were treated with different nitrate concentrations in
Arabidopsis thaliana.
 It was found that nitrate regulates many genes in central metabolic pathways such as the TCA
cycle. Responsiveness and nitrate regulation for all genes coding for TCA cycle enzymes were
analysed.
 Among the genes regulated by nitrate, a malate dehydrogenase gene (MDH, At3g47520), two
genes coding for NAD+ dependent isocitrate dehydrogenases (At5g03290 and At4g35260) and
a putative NADP+ dependent isocitrate dehydrogenase were found.
Source: The rules of gene expression in plants, Aceituno et al., 2008

11. ( Aceituno et al., 2008)

12. Regulation of transcription
 Specificity factors alter the specificity of RNA polymerase for a given promoter or set of
promoters, making it more or less likely to bind to them.
 Repressors bind to the Operator, coding sequences on the DNA strand that are close to or
overlapping the promoter region, impeding RNA polymerase's progress along the strand,
thus impeding the expression of the gene.
 Activators enhance the interaction between RNA polymerase and a particular promoter,
encouraging the expression of the gene.

13. In eukaryotes, gene activation occurs at a distance

14. Specialized cell types
 All cells must be able to switch genes on and off in response to signals in their environments.
But the cells of multicellular organisms have evolved this capacity to an extreme degree and
in highly specialized ways to form an organized array of differentiated cell types
(Determination).
 Cell Memory - prerequisite for the creation of organized tissues and for the maintenance of
stably differentiated cell types.
 Eukaryotic genes are regulated by combinations of proteins.
 Combinatorial control - the way that groups of regulatory proteins work together to determine
the expression of a single gene.

16. Continued
 General transcription factors that assemble at the promoter are the same for all genes
transcribed by polymerase II, the transcription regulators and the locations of their
binding sites relative to the promoters are different for different genes.
 The effects of multiple transcription regulators combine to determine the final rate of
transcription initiation.

17. Post-transcriptional regulation
 After the DNA is transcribed and mRNA is formed, regulation on how much the mRNA
is translated into proteins takes place.
 Modulating the capping, splicing, addition of a Poly(A) Tail, and sequestration of the
RNA transcript.
 Untranslated Regions of mRNAs - one of the most common ways of regulating how
much of its protein product is made is to control the initiation of translation.
 Eukaryotic mRNAs possess a 5’ cap that helps guide the ribosome to the first AUG.
 Repressors can inhibit translation initiation by binding to specific RNA sequences in the
5’ untranslated region of the mRNA and keeping the ribosome from finding the first AUG.
 When conditions change, the cell can inactivate the repressor and thereby increase
translation of the mRNA.

19. Regulation of translation
MicroRNA (miRNA)
 The 3'-UTR often contains miRNA response elements (MREs). MREs are sequences to which
miRNAs bind.
 By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of
various mRNAs by either inhibiting translation or directly causing degradation of the
transcript.
 The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the
expression of a mRNA.

21. Transposons
 Sequence of DNA which don’t have a fixed position on the genome.
 Produces transposae – causes movement of DNA.
 ACDS system in maize – Activator Disassociation system – reason for the grain colour
of maize – purple or yellow.
 AC – produces transposae – moves DS to target site C (purple colour) and inactivates it
producing yellow colour.

22. RNA Interference(RNAi)
 Targeted RNA degradation mechanism.
 Orchestrate the destruction of ‘foreign’ RNA molecules, specifically those that are
double-stranded.
 The presence of foreign, double-stranded RNA in the cell triggers RNAi by first
attracting a protein complex containing a nuclease called Dicer.
 Dicer cleaves the double-stranded RNA into short fragments called small interfering
RNAs (siRNAs).

24. miRNA Target Decoys
 miRNA target decoys are endogenous RNA that can negatively regulate miRNA activity.
 Engineering and precise manipulation of an endogenous RNA regulatory network through
modification of miRNA activity also affords a significant opportunity to achieve a desired
outcome of enhanced plant development or response to environmental stresses.
 By altering the sequence of the miRNA decoy sites, it is possible to attenuate miRNA
inactivation, which allows for fine regulation of native miRNA targets and the production of a
desirable range of plant phenotypes.
 The ability to inactivate a selected set of miRNAs allows regulation of multiple endogenous
miRNA targets coding for genes involved in related or unrelated processes and provides an
opportunity for complex trait engineering in plants.
Source: Regulation of Gene Expression in Plants through
miRNA Inactivation, Ivashuta et al., 2011.

25. References
 Bell JT, Pai AA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, Gilad Y, Pritchard JK
(2011). "DNA methylation patterns associate with genetic and gene expression variation in HapMap
cell lines". Genome Biology.
 Bird A (2002). "DNA methylation patterns and epigenetic memory". Genes Dev. 16(1): 6–21.
 Aceituno, F.F., Moseyko, N., Rhee, S.Y. and Gutiérrez, R.A., 2008. The rules of gene expression in
plants: organ identity and gene body methylation are key factors for regulation of gene expression in
Arabidopsis thaliana. BMC genomics, 9(1), p.438.
 Ivashuta S, Banks IR, Wiggins BE, Zhang Y, Ziegler TE, et al. (2011) Regulation of Gene Expression
in Plants through miRNA Inactivation. PLoS ONE 6(6).
 Palmero EI, de Campos SG, Campos M, de Souza NC, Guerreiro ID, Carvalho AL, Marques MM (Jul
2011). "Mechanisms and role of microRNA deregulation in cancer onset and progression". Genetics
and Molecular Biology. 34 (3): 363–70.