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Flashcards in Lecture 18 Deck (16):

Recap of gene silencing through siRNAs

- siRNA precursor is a long perfectly complimentary dsRNA molecule
- Dicer in the cytoplasm cuts it up into 20-24nt siRNA duplexes (a single siRNA precursor gives rise to multiple siRNA duplexes)
- Argonautes in the siRISC complex
- One strand of the duplex enter Ago, and this acts as a guide for RISC, and siRISC causes mRNA degradation.
- Post-transcriptional


Recap of gene silencing through miRNAs:

- miRNA precursor is a single miRNA per precursor, it contains an RNA hairpin loop, is not perfectly complementary
- The precusor arises by Drosher recognising in the nucleus, then dicer clips it in the cytoplasm to produce only one miRNA duplex.
- One strand of the duplex enter Ago, and this acts as a guide for RISC, and miRISC causes protein synthesis blockage.
- Post-transcriptional


How an activated RISC effects the transcript with slicing:

- The guide RNA in RICS is near perfectly complimentary and so it cleaves (with the help of Ago) the transcript.
- This is sequence specific and it slices the middle of the guide RNA (between residues 10 and 11).
- The transcript is degraded as unprotected ends are targeted by endonucleases.


How an activated RISC effects the transcript with silencing:

- The guide RNA in RISC is not perfectly complementary to the target transcript so it induces translational silencing.
- The formation of the 43S pre-initiation complex cannot occur
- Deadenylation of the plyA tail, occurs so this RNA is degraded
- this occurs in most animal miRNAs


miRNA target recognition in plants:

- Almost complete complementarity with target mRNA
- Target one gene/closely related members of a gene family
- Mainly transcription factors involve in developmental patterning or cell differentiation


miRNA target recognition in animals:

- Animal miRNA's do not slice because the sequence is not totally complementary
- Search for 3' UTR that are complementary to 8nt sequences
- Use genomic sequence to identify related genes and align 3' UTR
- Search within aligned 3' UTRs for occurance of conserved seed sequences


recognition in animals:

- one miRNA can have many targets
- one gene can be targeted by multiple miRNAs
- Targets are enriched for transcription factors involves in cell differentiation and physiological pathways
- There are hundreds of conserved targets per miRNA family


miRNA features:

- Work later in development to continue a cell type or initiate cellular differentiation
- They have precise spatiotemporal patterns of expression
- switch
- insulator
- tuner


Regulatory roles of miRNAs:

- Genetic switch, completely suppressing gene activity eg) temporal regulation of gene activity, spatial regulation of gene activity


miRNAs as a switch - spatial

- Target gene is active in tissue A but the promoter is also active in tissue B
- Target gene and miRNA are co-expressed in tissue B
- miRNA completely blocks protein synthesis


Blood development in zebrafish - use in situ hybridisation to look at the expression pattern of miR451 and target gata2:

- miRNA activity rapidly clears target gene activity from cells/tissues


Insulators of gene expression:

- Limit gene activity and reinforce transcriptional regulation
- Target gene is active in tissue B but the promoter is also occasionally active in tissue B.
- miRNA is only expressed in tissue B as it completely blocks protein synthesis
- Confers precision and robustness to gene expression


Loss of an insulator results in:

- the leaky transcript producing some protein
- the tissue may express something it is not supposed to


Functions of miRNAs, tuning of gene expression:

- Limit gene activity, controlling the gene activity in cells and tissues
- The target gene and miRNA are co-expressed in tissue B so miRNA partially blocks protein synthesis


miRNA acting as a tuner eg) in drosophila miR-8 and atrophin

- Atrophin is important for brain function
- It is expressed in both the larvae and adult
- it has 4 miR8 target sites in 3' UTR
- miR8 and atrophin are co-expressed
- miRNa ensures optimal gene activity in tissues
- If the tuner is lost miR8 displays cell death in the brain and morphological and behavioural defects


In order to distinguish how an miRNA is functioning:

- look at where it is expressed, and what effect it has on its target sequence