Post-translational regulation of gene expression

Question 1

Eukaryotic mRNA processing

Answer 1

Regulates gene activity
Provides diversity (alternative splicing)
Defective mRNAs are detected and degraded (quality control)

Question 2

Sequential recruitment of processing complexes

Answer 2

RNA polymerase II C-terminal domain

CTD partial phosphorylation during initiation recruits capping enzyme
Additional phosphorylation during elongation recruits splicing machinery
Transcription past 3’ end leads to recruitment of 3’ end processing complex

Question 3

5’ capping factors

Answer 3

5’ end capped with methylguanine nucleotide via 5’-5’ triphosphate linkage

RNA triphosphatase removes terminal phosphate at 5’ end
Guanyl transferase uses GTP to attach a guanosine monophosphate (GMP) to the end in 5’-5’ linkage
Guanine methylated by guanine-7-methyl transferase

Question 4

Splicing factor recruitment

Answer 4

CTD phosphorylation of ser 2 enhances spliceosome assembly

Question 5

Polyadenylation

Answer 5

Cleavage occurs after CA between a conserved AAUAAA hexamer and U/GU rich region

Poly(A) polymerase adds 200 adenosines after cleavage

Question 6

Alternative polyadenylation

Answer 6

Multiple polyadenylation sites in some mRNAs
At distal site, polyadenylation retains regulatory sequences
At proximal site, regulatory sequences are eliminated

Question 7

RNA splicing

Answer 7

Use transesterification reactions
All introns removed
5 snRNPs involved in splicing are U1, U2, U5, U4 and U6. Each has a snRNA and proteins.
snRNPs form the spliceosome

Question 8

Transesterification

Answer 8

Single phosphodiester bond is broken and replaced by another phosphodiester bond of similar energy (so no ATP required)

Question 9

Spliceosome assembly

Answer 9

snRNAs form base pairs with the pre-mRNA (recognition)
5’ splice site recognised by U1 and other sequences are bound by non-snRNPs
Rest of the apparatus binds, including branch-point binding protein
pre-mRNA rearranges, first transesterification occurs and lariat forms
Other rearrangement brings two exons together for second transesterification

Question 10

Splice sites

Answer 10

Defined by short sequence motifs
Recognised by the spliceosome
Introns usually have 5’-GU and 3’AG at their ends.

Question 11

Exon Junction Complex

Answer 11

Left at splice junctions after splicing
Marks transcript as processed and interacts with export/translation proteins
Also involved in nonsense-mediated mRNA decay (NMD). This recognises mRNA with premature termination codon and initiates their degradation to prevent harmful protein formation

Question 12

Recognition of correct splice sites

Answer 12

sequences defining intron-exon junctions can occur randomly (cryptic splice sites)
Exon definition enables spliceosome to recognise true splice sites.
Exon marked with SR proteins and introns bound with hnRNPs

Question 13

Alternative splicing

Answer 13

Specific splicing factor binding to intronic/extronic splicing enhancers promotes inclusion of alternative exon.

Binding of factors to splicing silencers inhibits splicing of alternative exon

Provides diversity in gene expression

Question 14

RNA mis-splicing and disease

Answer 14

cis-acting mutations in the core consensus sequences (5’ss and 3’ss)
Point mutation generating an alternative 3’ss in HBB gene (encodes beta-globin) results in beta+ thalassaemia. This is characterised by reduced beta-globin levels and anaemia

Question 15

Splicing and Alzheimer’s disease

Answer 15

Aberrant splicing of amyloid precursor protein and tau protein