RNA maturation

Question 1

RNA maturation def

Answer 1

processing of the primary transcript into the final product

Question 2

3 types of processing

Answer 2

1. cutting
2. splicing
3. chemical modification

Question 3

purpose of processing of different types of RNA

Answer 3

1. mRNA: regulatory role
2. rRNA and tRNA: improve functional and structural stability of molecule

Question 4

how is rRNA modified

Answer 4

-1 large transcription unit made up of operons repeated in tandem
-separated from non coding spacers (non coding regions between genes)
-cleavage of rRNA molecules and degradation of spacer regions

Question 5

how is tRNA modified

Answer 5

-removal of the 5’ leader sequence and 3’ trailer
-certain bases are modified
-addition of CCA sequence at the 3’ end of the molecule by enzymes
-Excision of the intron at the base of the molecule

Question 6

what are the differences in modification in prokaryotes and eukaryotes?

Answer 6

• EUK: all types of RNA modified to produce mature RNA

-PRO: undergo less/none modification because all protein synthesis process occurs in a very similar environment

Question 7

what are the features of a mature mRNA?

Answer 7

-5’ CAP
-polyA tail at 3’
-introns removed and exons annealed

Question 8

what is the purpose of mRNA cap and polyA addition?

Answer 8

-shows that the mRNA is in its mature version
-allows the cell to pass it from the nucleus to the cytoplasm to begin translation

Question 9

introns and exons definition

Answer 9

INTRONS: regions of the primary transcript that are removed from mature mRNA

EXONS: regions of the primary transcript that remain and are annealed to form the mature mRNA

!!!SOS: exons don’t necessarily all code for mRNA (eg. UTR regions) and vice versa for introns

Question 10

5’ capping process and functions of cap (4)

Answer 10

-catalysed by 3 enzymes that are bound to the CTD tail of RNAP2
-addition of a GTP cap backwards (meaning the 5’ end now ends in a guanine)

ROLES OF CAP:
1. prevents digest of mRNA via phosphatases
2. 1st recognition of ribosome in translation
3. permits binding of proteins that carry mRNA to cytoplasm
4. enhances splicing

Question 11

Polyadenylation process and role of poly A tail ()

Answer 11

-occurs right after mRNA cleavage by CPSF in termination
-around 250A added by the enzyme polyadenylate polymerase

ROLE:
-prevents mRNA degradation (by slightly extending the mRNA sequence)
- allows translocation into cytoplasm for trasnaltion

Question 12

splicing def

Answer 12

removal of introns and annealing of exons that forms the mature mRNA from primary transcript

Question 12

How are the steps of RNA maturation coordinated

Answer 12

using the phosphorylation of CTD RNAP2 tail:
-phosphorylated by kinase
-unphosphorylated by phosphatase

1. START: unphosphorylated tail
   2.phosphorylation induces capping
2. change in phosphorylation sites: induces polyadenylation
3. dephosphorylation: back to the start of cycle

!!! changes in the CTD residues phosphorylated occur at each stage with different kinase/phosphate combinations: allows coordination of steps

Question 13

discovery process of the existence of introns

Answer 13

1979: scientists observed that:

1. not all mRNA synthesised was translocated into cytoplasm
2. length of mRNA was shorter than the length of gene it was transcribed from

CONCLUSION: certain regions were removed from mRNA

Question 14

Structure of splicing junctions - how are splicing areas identified?

Answer 14

!! splicing point is determined by specific binding sites of consensus sequence:
From 5’ to 3’:

1. upstream exon
2. 5’ splicing site (GU start)
3. branch site (A base)
4. 3’ splicing site (AG end)
5. downstream exon

splicing junctions are recognised by splicing machinery for binding

Question 15

Spliceosome and function of snRNPs (3)

Answer 15

• large ribonucleoprotein that mediates splicing process
  -contains 150 proteins and uracil rich snRNPs (U1 to U6)

snRNP functions:
1. recognise 5’/branch/3’ site, bringing these sites in close proximity
2. allow hybridisation of mRNA with the splicing junction consensus sequence
3. catalysing RNA cleavage

SOS: snRNPs are small nuclear ribonucleoproteins

Question 16

SPLICING SEQUENCE

Answer 16

!! Made of 2 cutting and 2 joining reactions:

1st cut: attack of 5’ exon by the OH- on branch A site –> transfer of OH group and formation of a FREE 5’ EXON

1st joining: formation of lariat at A branch site due to self folding

2nd cut: attack of 5’ exon (using OH- group) to the phosphodiester vond between branch site and 3’ exon

2nd joining: 5’ exon annealing with 3’ exon

!!! introns are then completely degraded which releases energy used in the splicing synthesis reactions

Question 17

alternative splicing def

Answer 17

pre-mRNA spliced in different ways (different exons annealed in different combinations) so that a single gene code can code for multiple different proteins

Question 18

how is alternative splicing able to occur?

Answer 18

-consensus sequences found on 5’/3’ splicing sites can be either strong or weak
-silencers and enhancers also mediate splicing
-SR proteins (rich in serine and arginine) act as splicing regulators

Question 19

Role of SR proteins in splicing (3)

Answer 19

1. ensure accuracy and efficiency of constitutive splicing
2. regulate alternative splicing
3. certain SRs expressed in certain cell types control splicing in cell-type specific manners

Question 19

2 main reasons for recognition errors in splicing

Answer 19

1. difficult for spliceosome to identify exons within the vast amount of introns present (especially since exons are around 20 times shorter in length than introns)
2. splice site consensus sequences are quite loose

Question 20

RNA editing def

Answer 20

any process other than splicing which results in a change of RNA transcript such that it differs from sequence of template DNA

Question 21

2 types of RNA editing

Answer 21

1. Base modifications (eg. adenine to inosine, or cytosine to uracil and vice versa)
2. Insertion/deletion