Lecture 18

Question 1

Recap of gene silencing through siRNAs

Answer 1

• 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

Question 2

Recap of gene silencing through miRNAs:

Answer 2

• 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

Question 3

How an activated RISC effects the transcript with slicing:

Answer 3

• 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.

Question 4

How an activated RISC effects the transcript with silencing:

Answer 4

• 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

Question 5

miRNA target recognition in plants:

Answer 5

• 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

Question 6

miRNA target recognition in animals:

Answer 6

• 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

Question 7

recognition in animals:

Answer 7

• 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

Question 8

miRNA features:

Answer 8

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

Question 9

Regulatory roles of miRNAs:

Answer 9

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

Question 10

miRNAs as a switch - spatial

Answer 10

• 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

Question 11

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

Answer 11

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

Question 12

Insulators of gene expression:

Answer 12

• 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

Question 13

Loss of an insulator results in:

Answer 13

• the leaky transcript producing some protein

- the tissue may express something it is not supposed to

Question 14

Functions of miRNAs, tuning of gene expression:

Answer 14

• 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

Question 15

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

Answer 15

• 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

Question 16

In order to distinguish how an miRNA is functioning:

Answer 16

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