RNA processing During Eukaryotic Transcription

Question 1

Bacteria

Answer 1

-DNA lies directly exposed to cytoplasm, which contains ribosomes where protein synthesis takes place.
-As mRNA molecules are transcribed, protein synthesis can start immediately

Question 2

Eukaryotes

Answer 2

-Transcription takes place in nucleus, transcription (protein synthesis) takes place in cytoplasm
-Before RNA exits the nucleus for translation, it must go through several RNA processing steps

Question 3

The RNA processing steps occur

Answer 3

DURING the transcription of eukaryotic genes (into mRNA)

Question 4

mRNA processing learn as part of transcription

Answer 4

RNA transcripts that are destined to become mRNA molecules are processed in 3 ways in this order:
1- RNA capping (5’ end)
2- RNA splicing (5-3’ end)
3- RNA polyadenylation/tailing (3’ end)

These processing steps occur during transcription as parts of mRNA transcript become available

Question 5

RNA capping

Answer 5

RNA capping involves a modification of the 5’ end of the mRNA transcript (end that is synthesised first during transcription)

Question 6

RNA is capped by

Answer 6

Addition of an atypical nucleotide: a guanine G nucleotide with a methyl group attached (the methyl group is what makes it atypical)

Question 7

Capping occurs

Answer 7

After the RNA pol has produced approx 25 nts of RNA (I.e. long before it has completed transcribing the whole gene)

Question 8

RNA tailing/polyadenylation

Answer 8

-Provides newly transcribed mRNAs with specialised structure at 3’ end (or tail)
-at 3’ end of eukaryotic mRNA first trimmed by enzyme that cuts mRNA at particular sequence of nts
-A second enzyme the adds a series of repeated adenine (A) nts onto the cut end
-This is referred to as the Poly-A tail
-Poly A tail is generally a few 100 nts long

Question 9

Why cap and tail?

Modification of mRNA molecules by capping and polyadenylation serves a number of purposes

Answer 9

1: increases the stability of the mRNA molecule
2: Aids in export of mRNA from nucleus to cytoplasm
3: identifies mRNA molecule as mRNA
4: used by protein synthesis machinery as an indicator that both ends of mRNA present: message is complete

Question 10

RNA splicing

Answer 10

• only approx 5% of RNA are initially transcribed in nucleus ever reaches cytoplasm
  -Eukaryotic genes have their protein coding sequences interrupted by long non-coding
  -coding sequences = exons (expressed sequences)
  -Non-coding sequences = introns (intervening sequences)

Question 11

Eukaryotic and bacteria genes are

Answer 11

Organised differently

Question 12

Bacterial genes generally do not have

Answer 12

Introns (which is why splicing is not required) and contain mostly coding (exon) regions, whereas eukaryotic genes contain both introns and exons (and often mostly introns)

Question 13

The process of RNA splicing

Answer 13

-each intron contains a 3 short nt sequences that act as cues for its removal and are found at or near each end of the intron and are very similar for all introns

Question 14

The process of RNA splicing (continued)

Answer 14

a) 5’ splice donor site (5’ end intron: exon G-U)
b) 3’ splice acceptor site (3’ end of intron A-G exon)
c) Branch site: within the intron, about 30 nts upstream from the splice acceptor, has an AT rich region with atleast one A

Question 15

The spliceosome: structure and function

Answer 15

Made up of 5 different proteins
Proteins are known as SnRNPs [‘snurps’]

Question 16

The spliceosome is a complex of snRNPs (pronounced as snurps)

Answer 16

Which recognise the 3 sites in each intron and initiate the process of splicing

Question 17

SnRNPs are proteins combined with

Answer 17

A subtype of RNA known as small nuclear RNAs (snRNA) and there are 5 types of known SnRNPs (U1, U2, U4, U5, U6) that make up the spliceosome

Question 18

It is the snRNA in each of the SnRNPs

Answer 18

That form complementary base pairs with the 3 intron sites

Question 19

Within the spliceosome

Answer 19

U1 recognises the 5’ splice donor sites (at beginning of intron sequence) and U2 recognises the adenine branch site and it is known as the spliceosome complex A

Question 20

U3

Answer 20

Not involved in spliceosome but it does exist

Question 21

U4, U5 and U6 come together as a tri-snRNP and complex with U1 and U2 and is now known as Spliceosome complex B

Answer 21

This activated complex B helps the branched A site cut the 5’ splice donor site (I.e. intron-exon border) so that the cut 5’ end of intron becomes covalently linked to adenine nt to form branched structure, U1 and U4 then dissociate from the complex leaving U2, U5 and U6

Question 22

The free 3’ -OH end of exon sequence then reacts with

Answer 22

Start of next exon sequence at the acceptor site to join the 2 exons together leaving the intron lariat structure which dissociates along with the remaining SnRNPs

Question 23

Alternative splicing

Answer 23

The process by which exons can be reconnected in multiple ways to produce different mRNAs which can be translated into different proteins (protein isoforms)

Question 24

RNA splicing allows eukaryotes

Answer 24

To pack more information into every gene: increases coding potential of the genome. Therefore different proteins can be produced from the same gene

Question 25

Approx 80% of human genes

Answer 25

Undergo alternative splicing

Question 26

Human examples of alternative splicing

Answer 26

The first example (1981) is a gene encoding thyroid hormone calcitonin in mammalian cells. Pre-mRNA contains 6 exons, the calcitonin mRNA contains only exons 1-4 (no exon 5 and 6)

Question 27

A second example

Answer 27

Human factor VIII gene codes for protein factor VIII that functions in blood clotting pathway and mutations in this large gene are responsible for the most prevalent form of haemophilia (blood clotting disorder)

Question 28

The gene is 200,000 nucleotide base pairs with

Answer 28

26 exons

Question 29

When the introns are spliced out of the mature mRNA molecule

Answer 29

It is very small in size compared to the original large gene size