miRNA and siRNA (RNAi)

Short ppt on miRNA and siRNA

1. miRNA and siRNA and their application in crop improvement Submitted to: Dr. Arna Das Assistant Professor, Department of Genetics and Plant Breeding, BACA, Anand. Submitted by: 1. E. Vijaykumar 2. Vikram Suvatar 3. Sumit Parmar 4. Vivek Chauhan 5. Swapnil Baraskar Department of Genetics and Plant Breeding BACA, Anand

2. Contents: Introduction History Biosynthesis of miRNA Gene silencing by miRNA Biosynthesis of siRNA Gene silencing by siRNA Fate of the cleaved mRNA Difference between miRNA and siRNA Applications

3. Introduction: A  microRNA  (abbreviated  miRNA ) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression Mature miRNA molecules are partially complementary to mRNA molecules due to which they can downregulate gene expression It is reported that typical mammalian cell contains about 50,000 different miRNAs

4. Small interfering RNA  ( siRNA ), sometimes known as  short interfering RNA  or  silencing RNA , is a class of double-stranded RNA non-coding RNA molecules, 20-25 base pairs in length, interfering with the expression of specific genes with complementary nucleotide sequences with mRNA and then degrades it. siRNAs consist of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand, which form a duplex 19 to 25 bp in length

5. Fig: miRNA Fig: siRNA

6. History: The first micro RNA was discovered by a group led by Victor Ambros including Lee and Feinbaum It was described for the roundworm Caenorhabditis elegans (C.elegans) by them in 1993. But the term micro RNA was introduced in 2001 siRNAs and their role in post-transcriptional gene silencing (PTGS) were first discovered in plants by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England and reported in  Science  in 1999 Fig. Examples of miRNA stem-loops, with the mature miRNAs shown in red

7. Micro RNA (miRNA)

8. Biosynthesis of miRNA: Biosynthesis of miRNA involves various steps as following: Synthesis of long ssRNA molecule from template DNA using key enzyme RNA Polymerase II. Characteristic feature of this ssRNA molecule is its complementarity in the nucleotides of the strands

9. Due to this complementarity, this long ssRNA molecule, which indeed is unstable will fold around itself and form a stem-and-loop structure This structure is now called as pri-miRNA (100-120nt)

10. e) The first step is carried out by the enzyme Drosha, a member of the RNase III family of enzymes. Drosha makes two cleavages that cut the stem-loop f) This enzyme works together with an essential specificity subunit protein (called Pasha in some organisms and DGCR8 in others), and together these two proteins form an active Microprocessor complex g) It cuts pri-miRNA and makes it shorter (about 70nt) called as pre-miRNA

11. h) Now this pre-miRNA is transported from nucleus to cytoplasm with the help of the protein Exportin 5

12. i) In the cytoplasm, 2 nd cleavage reaction occurs using the enzyme Dicer which is a member of RNAse III family of enzymes j) Dicer cuts pre-miRNA at specific locations to cleave off hairpin to make double stranded RNA structure with overhanging pieces at both the ends

13. We get the miRNA-miRNA* duplex which is not fully complementary Out of these two strands, one is called guide strand and other is called passenger strand

14. Gene silencing by micro RNA: This duplex has two strands termed as guide strand and passenger strand. Passenger strand is removed using RISC i.e. RNA-induced Silencing Complex This processing of removing passenger strand is known as Sorting The central component of RISC is a protein called Argonaute Argonaute protein has a domain called PIWI which is a member of RNase H family. It will cleave one strand of target site

15. RISC finds the complementary sequence of miRNA on the target mRNA and when it is found, RISC binds to it. This binding process is known as nucleation It can work in in two ways, either by cutting the strand or by blocking translation by inhibiting the binding of ribosomal subunits to the mRNA If the miRNA complementarity with mRNA is quite high, then strand cleavage will occur and gene expression will be inhibited If the miRNA complementarity with mRNA is low, then it will block translation via inhibiting the ribosomal subunit binding to mRNA

16. Small interfering RNA (siRNA)

17. Biosynthesis of siRNA: In contrast to miRNA, which are synthesized in the cell using dsRNA formed endogenously, siRNA can be synthesized by using dsRNA obtained exogenously or endogenously Endogenous: By transcription of both strands of DNA to form dsRNA Exogenous: By the means of microinjecting dsRNA into the cell or by viral infections to the cell

18. Exogenous application of dsRNA into the cell: a) By viral infection b) By microinjection

19. Processing of this siRNA has to be done. For this purpose, a RNAse III enzyme known as Dicer helps in this process Dicer cuts the siRNA from both the sides to produce siRNA with 3’ overhanging with 2 nucleotides from both the sides

20. This 20-25 nucleotide long siRNA duplex is much similar to that of miRNA-miRNA* duplex. But the difference is the siRNA duplex is fully complementary while miRNA-miRNA* duplex is partially complementary siRNA duplex associates with RISC, which is composed of Argonaute protein

21. We get the siRNA duplex which is fully complementary Out of these two strands, one is called guide strand and other is called passenger strand

22. Gene silencing by small interfering RNA: Argonaute protein has a domain called PIWI which is a member of RNase H family. It will cleave one strand of target site As the strand complementarity of siRNA is higher, it is bit difficult to remove one strand as compared with case of miRNA So the passenger strand is removed and only guide strand remains attached to the RISC

23. Cleavage process

24. This complex is now ready to target the specific mRNA It searches for the specific target sequence in the mRNA and binds to it mRNA siRNA RISC

25. Argonaute protein cleaves the target mRNA at the binding site and prevents the expression of the gene i.e. gene silencing

26. Fate of the cleaved mRNA: The cleaved mRNA has two different fates: Degradation Adherence with another RNA strand to form dsRNA

27. Difference between miRNA and siRNA : Particulars miRNA siRNA Prior to Dicer processing Precursor miRNA with interspersed mismatches and hairpin structure Double-stranded RNA L ength 21-23 nucleotides 20-25 nucleotides Complementary Partially complementary to mRNA Fully complementary to mRNA mRNA target One Multiple Mechanism of gene regulation Translational repression Degradation of mRNA Endonucleolytic cleavage of mRNA (in case of very high complementarity only) Endonucleolytic cleavage of mRNA Clinical applications Drug target Therapeutic agent Diagnostic and biomarker tool Therapeutic agent

28. Applications in crop improvement: Gene silencing via miRNA and siRNA can be employed successfully to improve yield of crop and fruit plants by manipulating the basic agronomic traits of plant such as height, inflorescence, branching and size Knockdown of a gene  OsDWARF4  in rice resulted in shorter plants with erect leaf architecture leading to increased photosynthesis in the lower leaves. Such plant has potential for improved yields under dense planting conditions (Feldmann, 2006) Down-regulation of lignin genes like cinnamate 4-hydroxylase (C3H), shikimate hydroxycinnamoyl transferase (HCT) and 4-coumarate-coA ligase (4CL) in plants reduced total lignin content, improved accessibility of cellulases for cellulose degradation and increased dry matter degradability ( Hisano et al., 2009)  Suppression of GA 20-oxidase ( OsGA20ox2 ) gene resulted in semi-dwarf plants from a taller rice variety QX1

29. DET1  gene was specifically degraded in transgenic tomatoes with suppressed  DET1 , accompanied by an increase in the level of flavonoid and carotenoid Similarly, it has been utilized to increase the carotenoid content of rapeseed ( Brassica napus ) by down-regulating the expression of lycopene epsilon cyclase ( ε-CYC ) gene Xiong et al. (2005) introduced dsRNA targeting a single unit of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a gene of ethylene biosynthesis pathway in tomato and suppressed the expression of its gene. The rate of ethylene production was significantly inhibited in the ripened fruits of transgenic plants leading to prolonged shelf life Resistance to  Potato Spindle Tuber Viroid  ( PSTVd ) infection was achieved in transgenic tomato plants producing dsRNA against PSTVd sequences (Nora et al., 2009) In  Arabidopsis , miR393 was reported to repress auxin signaling by negatively regulating the F-box auxin receptors like  TIR1 , thereby restricting the infection by bacteria  Pseudomonas syringae  (Navarro et al., 2006) Suppression of a rice gene  OsSSI2 led to enhanced resistance to blast fungus  Magnaporthe grisea  and leaf blight bacterium  Xanthomonas oryzae  (Jiang et al., 2009)