RNA Processing in Eu genes

Question 1

What sequence is at the end of introns?

Answer 1

GUAGs

Question 2

Why is RNA capped at the 5’ end?

Answer 2

• Chem modified by addition of cap
• needs to be capped in order to mature
• provides barrier to 5’ exonuclease activity + stabilises transcript
• prevents degradation of fragile RNA e.g. by nucleotides on fingertips

Question 3

What is added during 5’ capping?

Answer 3

A 7-methyl guanosine (M7G) residue is added to the 5’ end of the new transcript.

So, GMP nucleotide added to new transcript in reverse orientation compared with normal 3’-5’ bridge

= now a 5’-5’ triphosphate bridge

Question 4

Which other RNA processing reactions does 5’ capping play a role in?

Answer 4

• splicing
• nuclear transport
• translation

Question 5

What does polyadenylation result in?

Answer 5

• stabilisation of RNA
• log polyA tail results in longer half life
• helps in nuclear export
• involved in translation

Question 6

Which proteins are involved in polyadenylation?

Answer 6

CPSFs - similar to TFs

Cleavage and poly-adenylation specificity factor

Binds to polyA site

Question 7

What is the poly(A) signal and what is the poly (A) site?

Answer 7

Site:
Where A’s get added

Signal:
AAUAAA

Question 8

What do CPSFs do?

Answer 8

Proteins which bind to polyA site on RNA

• have a high affinity for 3’ end/motifs
• binding causes a bend

Question 9

What happens after CPSFs bind to the polyA site?

Answer 9

1) at least 3 additional proteins bind (CstfF, cleavage stimulators factor, and cleavage factors 1 and II) = stays bent
2) PAP poly A polymerase binds to the complex and causes enzymatic cleavage - 10-35 nt upstream of the polyA signal where the bend is
3) the free 3’ end generated is polyadenylated (slowly) using ATP
4) cleavage factors are released, as is the downstream cleavage product

5) multiple copies of PABII (polyA binding protein) bind to the short A tail
- binding of PABII accelerates the rate of A addition by PAP (rapid polyadenylation)
- after 200-250 residues have been added, PABII signals PAP to stop polymerisation

Question 10

What is splicing?

Answer 10

Eu genes contain introns which are transcribed into hnRNA and need to be removed

Question 11

What corresponds to the start and end of biological info for a protein-encoding gene?

Answer 11

Start = initiation codon

End = termination codon

Question 12

What are the 2 ways RNA processing is regulated?

Answer 12

• differential splicing
• alternative splicing

= give rise to a series of mRNAs containing different exons and each specifying a different protein

Question 13

What is the main mechanism for regulating mRNA processing?

Answer 13

Alternative splicing

Common in human genome (>60% human genes are alternatively spliced)

Question 14

What are the general steps for processing eukaryotic genes?

Answer 14

1) transcription, 5’ capping
2) cleavage at poly(A) site
3) polyadenylation
4) RNA splicing

Question 15

What is the spliceosome?

Answer 15

Large, protein-RNA complex that consists of five small nuclear RNAs (U1, U2, U4, U5, and U6) and over 150 proteins

Question 16

What binds to specific sequences on the pre-mRNA substrate?

Answer 16

U1 and U2 snRNP complexes associate with pre-mRNA via base-pairing

• formed from snRNAs and their associated proteins

Question 17

How is a spliceosome made?

Answer 17

1) U1 and U2 associate with different exons on the pre-mRNA substrate
2) More snRNPs (U4, U5, U6) come and join the complex = spliceosome

Question 18

What are the steps in splicing?

Answer 18

1) All snRNPs join and form spliceosome

2) BP re-arranged between snRNAs = converts spliceosome into catalytically active configuration.
U1 and U4 are destabilised and released.

3) U2 and U6 (the catalytic core) catalysed the 1st TE reaction = forms intermediate containing 2’-5’ phosphodiester bond (proteins act like molecular scissors)

4) Further re-arrangements between snRNPs, the 2nd TE takes place:
Join 2 exons by a standard 3’,5’-phosphodiester bond + releases the intron as a lariat structure, and the snRNPs

5) The excised lariat intron is converted into a linear RNA by a debranching enzyme

PROTEINS MADE IN THR CYTOPLASM, COME BACK INTO NUCLEUS, BIND TO PRE-MRNA + HELP SPLICE EXONS OUT

Question 19

What is differential splicing?

Answer 19

25k genes in humans

Many mRNAs undergo alternative or differential splicing

= give rise to a series of mRNAs containing different exons and each specifying a different protein

Question 20

What are promoters, enhancers/silencers and insulators?

Answer 20

DNA sequences that regulate gene transcription

Question 21

What is alternative/differential splicing?

Answer 21

A regulated process during GE which results in a single gene coding for multiple proteins.

Within this process, particular exons of a gene may be included within or excluded from the final, processed mRNA, produced from that gene.

Question 22

What are splice junctions used for?

Answer 22

Differential splicing.

GUAG introns comprise >98% of splice junctions in the human genome.

Specific exons many be included or excluded in the RNA product by using or failing to use a pair of splicing junctions.

Selectively remove or include certain exons = change protein

Question 23

What are 2 examples of RNA processing?

Answer 23

1) differential splicing to produce different proteins: sex determination in drosophila
2) how polyadenylation is regulated: by blocking the polyadenylation step to reduce the amount of protein made e.g. U1A protein