LECTURE 5: Small RNAs in the regulation of biological processes

Question 1

what is the C value paradox?

Answer 1

• C value means the genome size - basically the complexity of an organism is not necessarily related to its genome size

Question 2

what proportions of DNA are conserved and junk? and what do each type do?

Answer 2

conserved DNA = 5% - conserved DNA is needed for protein coding - or needed for non coding RNA junk DNA = 95% - it is still transcriptionally active it just doesn’t code for proteins

Question 3

what is non coding RNA?

Answer 3

this is any RNA molecule that is not translated into a protein - (therefore this does not include mRNA )

Question 4

what is another name for non coding RNA? and what are examples?

Answer 4

• this category includes house keeping ncRNAs - mircroRNA : control of the translation of most genes - siRNA : this is needed for viral defence - piRNA : this is important for germ cell production - long ncRNA : needed for imprinting and gene regulation (related to Xist and H19 and IGF-1)

Ms Peggy Lipton

Question 5

how is antisense RNA used as a tool to block the function of mRNA?

Answer 5

• if there is a gene you want to block - you can transcribe the reverse of the gene to make there antisense strand RNA - the antisense stand and sense strand make hydrogen bonds together - this is basically a double stranded RNA - this means the sense RNA stand cannot be translated and double stranded RNA is made which is basically a silencing trigger

Question 6

why might RNA silencing be important?

Answer 6

• might be important because some plants use it as a defence mechanism against a viral infection

Question 7

what is used to make siRNA and what does siRNA do?

Answer 7

• double stranded RNA is used to produce siRNA - si RNA inhibits the RNA that makes the double stranded RNA

Question 8

how does the si RNA work?

Answer 8

• DICER (this is a gene) breaks up dsRNA into 21- 25 bp fragments - DICER works like an enzyme - passenger strand is removed - the required protein is argonaute piwi proteins (AGO) - the remaining strand is the antisense to the target strand - RNA induced silencing complexes are made and these recognise and cleave target mRNA molecules which have complementary sequences to the single stranded guide si RNA

Question 9

why is DICER an essential gene?

Answer 9

• without dicer there would be lethality in early stage embryonic stages due to embryonic stem cells not being able to differentiate - there would a depletion of multipoint stem cells -

Question 10

why is si RNA able to enter cells? why is this therefore important?

Answer 10

DNA fragments that are 25bp or smaller can get into cells without causing detrimental cytokine reactions si RNA is smaller than that therefore can get into the cell without invoking a cytokine response therefore siena can be used as a therapy to turn off genes that we don’t want to be expressed

Question 11

* how to make si RNA?

Answer 11

can be synthesised artificially it can be synthesised as one long piece of RNA that can fold back on itself = small hairpin RNA

Question 12

why does genetically encoded mean?

Answer 12

• this means it occurs naturally within genes

Question 13

what was found out in the investigation about the action of Lin -14 (protein)

Answer 13

• RNA production for Lin 14 persists throughout development - the protein production for Lin 14 disappears pretty early on in development - there are other mutants which are not lin -14 mutants but can give the same phenotype - another set of genes was found called lin -4 which controlled the loss of protein - however when lin 4 was investigated no genes were found - instead it was found that lin 4 was a micro RNA which can bind to Lin 14 mRNA to stop it from making proteins

Question 14

what is the main purpose of miRNAs like Lin -4

Answer 14

they are needed to regulate other genes, it is another level of gene regulation

Question 15

how are miRNA’s transcribed and processed?

Answer 15

• miRNAs are initially made as pri-mRNAs - the pri - miRNAs are then processed and chopped up into pre - miRNA where it is then transported in the cytoplasm - the pre -miRNA is then passed down into the RISC complex - the pre - miRNA can then target genes at the RNA level and silence genes

Question 16

what is gene knockdown?

Answer 16

this is decreasing the amount of protein you get from a gene

Question 17

* what do the mi -RNA genes look like?

Answer 17

there are two matching areas which are separated by a bulge. therefore looks like match - bulge - match - it can also fold over to form the shRNA

Question 18

what is the seed region?

Answer 18

• this is the most important region of the miRNA for targeting - it lies between the nucleotide positions 2 to 8 from the 5’ end and it is often flanked by adenosine

Question 19

explain what happens to the micro RNAs in chronic lymphoid leukaemia?

Answer 19

• the delation of part of the gene on chromosome 14 leads to the loss of miRNA and promotes chronic lymphoid leukaemia

Question 20

how might chromic lymphoid leukaemia be treated?

Answer 20

by supplying miRNA the cancer might be destroyed