mRNA processing

Question 1

When does mRNA processing occur?

Answer 1

Cotranscriptional, occurs at same time

Question 2

Order of mRNA processing

Answer 2

Capping
Splicing
Polyadenylation

Question 3

What is mRNA called directly after transcription?

Answer 3

pre-mRNA

Question 4

Where does mRNA processing occur?

Answer 4

In the nucleus

Question 5

What happens to G residue in capping?

Answer 5

Methylated and binds to protein complex called cap binding complex (methyl guanosine cap)

Question 6

What are the roles of 5’ capping (4)?

Answer 6

Protect 5’ of mRNA from degradation by exonucleases

Improve ribosomal recognition for translation

Provide scope for translation regulation

Recognised by export machinery - helps mRNA move out of nucleus

Question 7

What is the 5’ cap?

Answer 7

Altered nucleotide on 5’ end

Question 8

What happens in capping?

Answer 8

GMP nucleotide/ G residue added to 5’ end of pre-mRNA by 5’-5’ triphosphate bond

Question 9

What is unique about the 5’ end linkage?

Answer 9

5’-5’ bond

Question 10

What does splicing do to exons and introns?

Answer 10

Excision of introns and fusion of exons

Question 11

What directs splicing?

Answer 11

Splicesome

Question 12

What does the spliceosome consist of?

Answer 12

5snRNAs bound to 100 splicing factors - forms complexes known as snRNPs

Question 13

What sort of reaction occurs in splicing?

Answer 13

snRNA mediated transesterification reaction

Causes 2 sequential breakages and rejoining of RNA sugar phosphate backbone

Question 14

Where are splice sites?

Answer 14

5’ and 3’ ones at border of each intron/exon

Question 15

What group is at the end of a 5’ cap , why is this important?

Answer 15

3’OH, not recognised by exonucleases

Question 16

What do snRNAs do?

Answer 16

Complementary sequence to splice site

Recruits components of spliceosome - RNA protein interactions between spliceosome and preRNA allows introns to be spliced

Question 17

What is the process of splicing?

Answer 17

2’ OH of adenosine in intron (branch point) nucleophilic attack on 5’ exon/intron border

Transesterification reaction causes lariat intron joined with 3’ exon and release of 5’ exon

3’ OH of 5’ exon attacks lariat intron/3’ exon junction

Transesterification then fuses 5’ and 3’ exons.

Question 18

What is found at the branch point?

Answer 18

2’OH of an adenosine

Question 19

What are snRNPs?

Answer 19

Small nuclear RNAs (snRNAs) bound to specific proteins

Ribonucleoproteins snRNPs

Question 20

What is at 5’ end of intron?

Answer 20

GU

Question 21

What is at 3’ end of intron?

Answer 21

AG

Question 22

What happens at exon exon junctions after splicing?

Answer 22

Marked by deposition of exon junction proteins/complex

Question 23

What is nonsense mediated decay?

Answer 23

Surveillance mechanism in which exon junction proteins act as markers to identify faulty mRNA

Question 24

How does nonsense mediated decay protected mRNA?

Answer 24

Exon junction complex downstream of stop codon recognised

Indicates premature stop codon so faulty mRNA degraded

Question 25

After what point should you no longer find exon junction complexes?

Answer 25

After normal stop codon.

Question 26

What does alternative splicing allow?

Answer 26

Tissue specific gene expression, several proteins to be made from single gene

Question 27

What correlates with increased complexity of organism?

Answer 27

More introns per gene and thus greater expansion of proteome.

Question 28

When/where can alternative splicing occur?

Answer 28

In different cell types or response to different signals

Question 29

How can alternative splicing be made to include certain exon?

Answer 29

Enhancer proteins bind enhancer sequence in exons, make splice site more attractive. More likely to include the exon in mRNA

Question 30

How can alternative splicing prevent splicing of a gene?

Answer 30

Proteins bind to silencer region prevent spliceosome associating with splice site (weaken splice site)

Question 31

What happens when enhancer proteins associated with mRNA?

Answer 31

Splicing factors more likely to recognise 3' and 5' splice sites that flank an exon

Question 32

How can an exon be skipped?

Answer 32

Negative factors bind specific exon sequences prevent recognition of 3' and 5' splice sites flanking exon

Question 33

What are 2 steps of polyadenylation?

Answer 33

premRNA cleaved 20 bases downstream by endonucleases Poly(A) polymerase adds Poly(A) tail to 3' end

Question 34

What does polyadenylation consensus sequence signal (AAUAAA)?

Answer 34

Signals cleavage of premRNA 20 bases downstream Signals polyadenylation

Question 35

What are cleave and polyadenylation machinery associated with?

Answer 35

AAUAAA and DSE sequence (second signal after cleavage site)

Question 36

How does polyA tail form

Answer 36

Poly(A) polymerase adds poly(A) tial to 3' end Donated by ATP which is then covered by Poly(A) binding proteins

Question 37

How many residues is poly(A) tail?

Answer 37

250 adenosine resiudes

Question 38

What donates adenosine residue for poly (A) tail?

Answer 38

ATP

Question 39

What happens after poly A tail formed?

Answer 39

Covered by poly(A) binding proteins

Question 40

What enzyme catalyses poly(A) tail formation?

Answer 40

Poly(A) polymerase

Question 41

What are the roles of Poly(A) tail (3)?

Answer 41

Uniform 3' end for nuclear cytoplasmic transport, stability, translatability Makes mRNA more stable because must be degraded by exonucleases before they reach coding mRNA, so more time for this to be translated Cap and poly (A) binding proteins interact, forming structure that recruits ribosome to mRNA- enhances translation

Question 42

How can mutations affect splice sites?

Answer 42

Lead to reduced/abolished splicing of some introns, or faulty mRNAs that contian intronic sequences.

Question 43

What can mutations affecting poly(A) signals do?

Answer 43

Increase/reduce cleavage and polyadenylation so total ouput of mRNA affected (i.e. poly(A) machinery may not recognise AAUAAA sequence)

Question 44

How can mRNA presence in the cell be regulated?

Answer 44

Transcription of siRNAs and miRNAs that target and break down mRNA in the cytosol.

Question 45

How do siRNAs break down mRNA?

Answer 45

siRNA originates as a long RNA strand that is double stranded. Dicer enzymes then catalyse the production of siRNAs, enabling the siRNA to associate with proteins to form a RISC complex that is single stranded. The siRNA then binds to the target mRNA and induces cleavage. So it cannot be translated.

Question 46

How do miRNAs break down mRNA?

Answer 46

Less specific short RNA sequences that bind to mRNA strands to physically block it from translation.