VL 6: RNAi Flashcards
Definition RNAi
RNA Interference:
Silencing of gene expression due to the action of short RNA molecules (21-24 nt).
It broadly refers to gene silencing that is triggered by some kind of small RNA in association with a member of the Argonaute family of proteins
RNAi Pathway can act on two levels:
1) TGS: Transcriptional gene silencing
2) PTGS: Posttransciptional gene silencing
How does RNAi work?
- Required: (long) double- stranded RNA molecule (dsRNA)
- dsRNA is processed by DICER into short RNA (siRNA, 20-25 nt)
- siRNA associate with ARGONAUTE effector complex (RISC)
- Complementary mRNA (sequence specificity!) is bound by RISC and inhibited
Where does the dsRNA originate?
- Biderictional transcrition (siRNA, Dicer)
- Inverted repeat transcription (siRNA, Dicer)
- Aberrant transcription (siRNA, Dicer)
- Transcription of primary piRNA clusters (piRNA, Piwi Protein)
Aberrant transcription refers to errors in the transcription process that lead to the production of incorrect or unintended RNA transcripts.
What are piRNA (PIWI-associated RNA)?
- metazoan germline
- function to silence transposons
- produced from long single stranded RNA polymerase II (Pol II) transcripts and have extensive sequence complementarity with dispersed transposons.
- Generation and amplification of piRNA: process involves Piwi clade of Argonaute proteins and their slicer activities (Aubergine and AGO3) – (not DICER and RdRP!)
What is DICER?
What is RISC?
DICER processes dsRNA into siRNAs (20-25 base pairs). In plants, the orthologues of DICER are called DICER-LIKE proteins.
RISC: Argonaute Protein binds siRNAs and inhibit their target mRNAs (forming the RISC)
-> cleave mRNA, inhibit translation or mediate transcriptional silencing.
(Arabidopsis has 10 Argonaute genes and four dicer-like genes
Different types of RNAi in plants
- PTGS: RNAi acts in PTGS when there are aberrant or excessive RNA transcripts present in the cytoplasm, such as those produced by overexpressed genes, viral RNA, or transposons.
- TGS: RNAi acts in TGS when aberrant transcription occurs, such as the faulty transcription of heterochromatin regions, including transposons and repetitive elements.
- microRNAs:! Regulation of endogenous processes in development and environmental response
1&2: Natural function of RNAi: Protecting the genome from virusus and transposon
If transposon RNA escapes TGS and is present in the cytoplasm, RNAi can trigger PTGS to degrade these transcripts.
Silencing of transgenes
Transgenes introduced into plants are frequently silenced by the siRNA pathway.
Silencing can be triggered by:
* Very high levels of gene expression
* dsRNA derived from transgenes
* Aberrant RNAs encoded by transgenes (single stranded and because of error in transcription oder sowas)
Transgenes are silenced post-transcriptionally and transcriptionally
Experiments to modify flower color in petunia gave early evidence of RNA silencing.
Manipulation of chalcone synthase expression to modify pigmentation (Petunia)
Wild-type petunia producing purple anthocyanin pigments,
Chalcone synthase (CHS) = enzyme at the start of anthocyanins biosynthesis
Hypothesis: sense RNA production enhances pigmentation and antisense RNA production blocks pigmentation.
But: both sense and antisense gene constructs led to the inhibition of pigment production, resulting in white or variegated flowers instead of deep purple.
-
-> Co-Suppression Phenomenon:
Introduction of extra copies of the chalcone synthase gene (either sense or antisense) triggered RNA interference (RNAi), leading to the degradation of both the introduced and endogenous chalcone synthase mRNA. - Conclusion: This revealed that the RNAi mechanism can be activated by the presence of both sense and antisense RNA, causing gene silencing and demonstrating the powerful regulatory role of RNAi in gene expression.
Co-supression
Co-suppression is the process where the introduction of additional copies of a gene leads to the simultaneous silencing of both the introduced gene and the endogenous gene.
It occured when overexpressing of the gene encoding CHS was attempted in petunia and this unexpectedly resulted in variegated or even completely white flowers instead of dark purple flowers.
-> because of coordinated gene silencing of both genes (the CHS transgene and the endogenouse CHS gene)
It is a consequence of siRNA production = RNAinterference
Transposons
- Fragments of DNA that can insert into new chromosomal locations (jumping genes)
- Some copy themselves and increase in number within the genome
- Responsible for large scale chromosomal rearrangements as well as single-gene mutagenic events
- powerful endogenous mutagenes that can impact genes and genomes in multiple ways e.g. cause inactive or unstable alleles
- highly abundant and found in all organisms
discovered in Zea mays by Barbara McClintock
Trasposons insertion can change patterns of tissue specific expression. Name two expamples
- transposon in tb1drove maize domestication: ‘Hopscotch’ variant swept to high frequency in modern maize populations, yield plants with few branches. -> transposable element (Hopscotch) inserted in a regulatory region of the maize domestication gene, teosinte branched1 (tb1), acts as an enhancer of gene expression
- Insertion of ‘Rider’ causes cold dependent expression of Ruby in fruit of blood oranges
Biological functions of RdDM
- TE Silencing & genome stability (gene inactivation or activation)
- Development & Reproduction (flowering time)
- Short & long- range signaling (cell-to-cell or systemic)
- Stress Response (heat or viral stress)
siRNA - what is their role as Genomic Defenders
- siRNAs protect the genome by Suppressing invading viruses, Silencing sources of aberrant transcripts, Silencing transposons and repetitive elements
- siRNAs are produced from transposons and repetetive DNA -> by the action of Dicer(-like) proteins dicing dsRNA into 24 nt siRNAs
- siRNAs associate with AGO proteins and form silencing complexes (RISC)
- These silencing complexes can act post-transcriptionally on RNA targets, cleaving them or interfering with translation
- The silencing complexes can also act on chromatin, silencing their targets by DNA methylation or histone modification (RdDM).
