lecture 7

Question 1

What does pre-mrna need to go through to be functional mrna?

Answer 1

it needs to be processed via splicing of introns

Question 2

When will RNA not be transcribed?

Answer 2

when its in a functional state

Question 3

What is the cap and polyA tail?

Answer 3

specific to eukaryotic mRNA
added post-transcriptionally to protect RNA from degradation to give stability and protection

Question 3

Why does RNA polyermase II have a long conserved tail?

Answer 3

to recruit factors involved in stability and protection

Question 4

What is the structure of the 5’ m7G cap?

Answer 4

All RNA polymerase II RNAs
RNA initially contains triphosphate at 5’ end between two bases
Second nucleotide in different orientation (added 5’ to 5’)
Methyl group added to top of base to change chemical behaviour
Capping two structure event: GpppN structure, methylation

Question 5

What are the functions of the m7G cap?

Answer 5

Protects mRNA from degradation by 5’-3’ nucleases
Facilitates splicing
Facilitates export from the nucleus
Critical for translation of most mRNAa
Functions mediated through protein binding - CBP80/CBP20 in nucleus (processing/export), elF4 complex in cytoplasm (translation) - caps can bind to proteins for export of RNA to cytoplasm where it binds to eIF4 complex

Question 6

What are the two key functions of capping?

Answer 6

mRNA protection and mRNA export

Question 7

True or False?
m7G cap is encoded in the genome

Answer 7

False - added post-transcriptionally

Question 8

What is the process of adding a polyA tail called?

Answer 8

polyadenylation

Question 9

In mammals are introns or exons usually longer?

Answer 9

introns - generally between 80-10,000 nucleotides long

Question 10

What are the conserved sequences in introns?

Answer 10

5’ splice site
3’ splice site
branch site

Question 11

Why do intron and exon boundaries contain conserved sequences?

Answer 11

sequences define limits of exon and intron
sequences recruit the splicing machinery required to remove the intron and join the exons

Question 12

What is the two step splicing process of introns?

Answer 12

1) cut at 5’ splice site, creation of bond between 5’ end of intron and branch site
2) cut at 3’ splice site to release intron lariat, ligation of two exons

Question 13

What is a spliceosome?

Answer 13

enzymatic complex that catalyses intron-exon splicing in eukaryotes requiring ATP
Proteins include: RNA-binding proteins, ATPases, GTPases
-five small nuclear ribonucleoproteins (snRNPs)

Question 14

What are small nuclear ribonucleoprotein particles (snRNPs)?

Answer 14

Stable RNA - protein complex in the nucleus
Non-coding RNA
RNAs base-pair with conserved sequences in the intron (U1 with the 5’ splice site, U2 with the branch point)

Question 15

What is splicing catalysed by?

Answer 15

snRNAs

Question 16

What is anti-Sm?

Answer 16

anti-sm antibodies react against sm proteins
very rare antibodies unless you have the autoimmune disease lupus erythematosus

Question 17

Why are transcription and splicing functionally coupled?

Answer 17

so that they influence one another

Question 18

How many conserved sequences are involved in splicing?

Answer 18

three

Question 19

what are snRNPs?

Answer 19

Rna protein complexes

Question 20

True or False?
Splicing is an RNA catalysed event

Answer 20

true

Question 21

How can splicing expand the proteome?

Answer 21

many genes have multiple introns
number of proteins&raquo_space; genes in the genome

Question 22

What are the different types of intron splicing?

Answer 22

Exon skipping
Intron retention
Mutually exclusive exons
Alternative 5’ splice site
Alternative 3’ splice site

Question 23

How are alternative splicing mechanisms regulated?

Answer 23

Regulated cis-acting sequences in pre-mRNA
Activators - bind to intronic and exonic splicing enhancers (ISE and ESE)
Repressors - binds to intronic and exonic splicing silencers (ISS and ESS)

Question 24

What mutations cause defects in splicing?

Answer 24

normal protein -> loss of normal function (NMD, protein instability, truncations)
two protein isoforms -> only one protein isoform

Question 25

What illnesses are caused by mutations in splicing?

Answer 25

Spinal muscular atrophy - most common genetic cause of infant mortality
Retinitis Pigmentosa - reduced visual capabilities and blindness
Myotonic Dystrophy - a muscle wasting disease