Untitled Quiz

Molecular Biotechnology BE618 Lecture 7: Gene Silencing

• Gene silencing occurs at both post-transcriptional (PTGS) and transcriptional (TGS) levels.
• PTGS, also known as RNA interference (RNAi), downregulates gene expression after transcription.
• Examples of PTGS include transposons, retroviral genes, and heterochromatin.
• Antisense technology, used since ~1980, aims to repress specific genes by introducing an antisense gene.
• Antisense technology's effectiveness is variable, often mediated by RNAi rather than translation inhibition.
• Co-suppression, a form of PTGS, was observed when an introduced gene with homology to an endogenous gene in petunia led to suppression of both genes.
• Co-suppression results mainly from degradation of mRNAs from both the introduced and endogenous genes.
• Genes involved in the flavonoid/anthocyanin pathway, such as CHS (chalcone synthase) and DFR (dihydroflavonol reductase), are affected by this process.
• Introducing these genes into petunia with a strong viral promoter led to decreased mRNA and pigment levels in transgenics.
• The 2006 Nobel Prize for Physiology or Medicine was awarded for RNA interference discovery in C. elegans (Andrew Fire & Craig Mello).
• RNA interference (RNAi) was initially discovered in C. elegans while attempting to use antisense RNA in vivo.
• "Sense" RNAs, when contaminated with dsRNA, also demonstrated gene suppression.
• Double-stranded RNA (dsRNA) induces mRNA interference more effectively than antisense mRNA.
• Suppression signals can move between cells.
• RNAi occurs in various eukaryotes, including angiosperms, chlamydomonas (unicellular), mammals, C. elegans, Drosophila, and Neurospora, but not in yeast.

RNAi Pathway

• Long double-stranded RNA (dsRNA) is processed in the nucleus, creating shorter dsRNA.
• This shorter dsRNA is exported to the cytoplasm, binding to and being cleaved by dicer into small fragments (siRNAs or miRNAs).
• siRNAs or miRNAs are recruited by the RISC complex, unwinding and incorporating into a protein-RNA complex.
• The RISC-associated siRNA or miRNA binds to a complementary mRNA molecule.
• The mRNA is then cleaved and degraded, preventing protein synthesis of the target gene.

Mechanism of RNAi: Role of Dicer

• Cells undergoing RNAi contain small fragments of suppressed RNA.
• A nuclease called Dicer, purified from Drosophila embryos, is involved in processing these small RNA fragments.
• Mutating the Dicer gene inhibits RNAi.
• Dicer (an endonuclease) degrades dsRNA into 21-24 nt fragments.
• In higher eukaryotes, Dicer separates the fragments via intrinsic helicase activity.

Model for RNAi

• RNAi is a very efficient process, generating many small interfering RNAs (siRNAs) from a larger dsRNA.
• It uses Dicer or an ATP-dependent helicase to cleave and separate dsRNA strands.
• Cleaved RNA fragments form active siRNA complexes called RISC (RNA-induced silencing complex), which contain Argonaute instead of Dicer.
• siRNA complexes target cleavage of mRNA, inhibiting protein synthesis of the target gene.

RNAi Mechanism

• RNAi mechanism involves the use of RNA-dependent RNA polymerase (RDR).
• In plants, fungi, C. elegans, and Drosophila, RDR participates in RNA silencing initiation or amplification.
• Initiation or amplification of silencing requires RDR access to the RNA template.
• RDR access is sometimes blocked by proteins like CBP (cap-binding protein) and PABP (poly(A)-binding protein).
• The process can initiate or amplify RNA silencing by producing dsRNA, which then gets further processed into smaller siRNAs.

Why RNAi Silencing?

• RNA interference evolved to protect genomes from viruses and potentially transposons/mobile DNAs.
• Some viruses have proteins (e.g., HCPro, P19, Tat) that suppress silencing to facilitate viral replication.

MicroRNAs (miRNAs)

• Very small (micro) miRNAs have recently been discovered in plants and animals.
• They are similar to siRNAs, regulating gene expression through mRNA degradation or translational repression.
• miRNAs are used for more than just defense.

Discovery of microRNA in 1993 and Nobel Prize 2024

• Researchers, including Victor Ambros and Gary Ruvkun, discovered microRNAs (miRNAs) important in the development of the roundworm C. elegans.
• The lin-4 and lin-14 gene mutations in worms reveal the crucial role of miRNAs.
• miRNAs are encoded by their own genes, are processed differently and have similar function to siRNAs, but are endogenous (within the organism).

C. elegans and heterochronic gene lin-4

• C. elegans lin-4 gene encodes small RNAs with antisense complementarity to lin-14.
• These small RNAs play a critical role in regulating gene expression during development.

GLO1 role in pollination

• GLO1 is essential for successful pollination in plants.
• RNAi-mediated silencing of GLO1 in plants leads to reduced seed set and increased methylglyoxal levels.
• Transgenic GLO1 RNAi lines show reduced pollen attachment and germination.

Comparison of miRNA Biogenesis and Action

• The mechanisms of miRNA biogenesis and action differ slightly between plants and animals.
• Plants primarily use DCL1 (Dicer-like 1) for miRNA processing.
• Animals utilize Drosha and Dicer.

Summary of differences between plant and animal miRNA systems

• Plant and animal miRNA systems share some similarities, but also have key differences in miRNA gene number, location within the genome, miRNA biosynthesis mechanisms, site of miRNA targeting within a gene, and function of known target genes.