Question

1. How is RNA interference mediated within cells?

2. Compare and contrast the origin, structure, function of miRNA and siRNA.

3. What are the applications of siRNA in basic research? In clinical research?

Please answer all questions. Thank you

Answer 1

Answer 1:

RNA interference (RNAi) is an emerging technique which is used in molecular biology to study the gene functions and host-pathogen interactions. Waterhouse discovered RNA interference (RNAi) based resistance in transgenic tobacco plants against Potato virus. RNA silencing mechanism participates in both biotic and abiotic conditions in plant defence against viral pathogens that help mankind to face the challenges of unfavourable environmental conditions. This application creates disease resistance in plants by suppressing a specific gene or genes. This mechanism would be operative only when the damaging virus is immensely alike to the transgene.

Genome replication within the plant cells and their systemic movement through plasmodesmata only possible by suppressing the RNAi mechanism. RNAi mechanism is based on post-transcriptional gene silencing system (PTGS), in which viruses from almost all the plant virus genera encode suppressors of RNA silencing (VSRs) which suppressed the key crucial stages of cellular RNAi system in host defence. RNA mediated virus resistance also called homology dependency resistance. In addition to repressing the host defence system via RNAi mechanism, these VSRs also helps in viral replication, movement of the virus, helps in encapsidation of viral nucleic acid and pathogenesis.

VSRs suppress RNA silencing mainly by two pathways: i). siRNA mediated RNA interference and ii). microRNA mediated RNA interference.

Answer 2:

siRNA:

An enzyme dicer plays a major role in siRNA mediated RNA interference pathway; it will convert the ds DNA into siRNAs duplexes of two approximately 21 nucleotides with the overhang of 2 nucleotides at 3’ end. It is a multidomain protein of approximately 200 kD, it belongs to enzyme family RNase III which functions in the conversion of dsDNA to siRNA. Dicer is having dsDNA binding domain, ATPase/ helicase domain and RNA III domain with catalytic activity. All these three domains comprise of RNA-induced silencing complex (RISC) loading complex. Hence dicer loads the RNA duplex into RISC. Here only one strand will direct the gene silencing called a guide RNA. Another strand called anti-guide strand get destroyed during activation of RISC. Argonaut proteins having endonucleases activity are the active components of RISC, they cleave the target mRNA molecule bound to siRNA.

miRNA:

miRNA highly conserved 20-25 mer RNAs which regulates gene expression post-transcriptionally via binding to the 3’ UTR of specific mRNAs. An enzyme Drosha which belongs to the family of RNase III enzyme works as a dsRNA specific endonuclease which will convert the pri-miRNA to hairpin RNAs of 70-100 nucleotides in length called pre-miRNA. Pre-miRNA is then transported to the cytoplasm. In the cytoplasm, dicer coverts the pre-miRNA duplex and load it into the RISC.

Gene silencing can be done either by mRNA degradation or preventing mRNA from being translated. miRNAs function forming base pairs with complementary sequences in target mRNA molecules. In case of complete complementarity in between the miRNA and target mRNA sequence, Argonaute will cleave the mRNA and result in direct mRNA degradation. But in case there no complete complementarity then the silencing is carried out by preventing the mRNA molecule being translated.

siRNA and miRNA

The small RNAs that provide target specificity to the silencing machinery-siRNA and miRNA can be distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly based-pair RNA, while miRNA originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNA were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA however, an endogenous source of siRNA have now been recognized. miRNAs were discovered through their critical roles in development and cellular regulation. miRNAs have always been recognized as being of endogenous origin.

Answer 3: siRNA is used for target validation, pathway analysis, drugs development, gene redundancy, and functional screening.