Post-transcriptional RNA Modifications

Gene Expression Part 2

Post-transcriptional Modifications

• Processing – cleavage of 45S rRNA precursor, base modifications of tRNAs, rRNAs

• Capping – mRNAs & snRNAs

• Polyadenylation - mRNAs

• Splicing – mRNAs, some tRNAs

Promoter

Transcription

Cleavage(the light pink regionsare degraded)

45S transcript

18S 5.8S 28S

5 318S 5.8S 28S

18S 5.8S 28SrRNA rRNA rRNA

Processing of 45S rRNA Precursor

35Endonuclease

Exonuclease(RNase D)

P = PseudouridineT = ThiouridineI = Inosine

mG = Methylguanosine

RNase P

mG TT

A

P

P

I

CC

Anticodon

Processing of pre-tRNAs

snoRNAs

Yeast C/D box snoRNA

D’ box

C’ box

Post-transcriptional Modifications Regulate Gene Expression

• Capping – Stability– Translation efficiency

• Polyadenylation– Stability– Translation efficiency– Timing of translation/storage of mRNA

• Splicing– Production of varied proteins from same gene– Developmental regulation – tissue specificity, sex

determination

Figure 12.19

Removes the -phosphate

Capping – Step 1

Capping –Step 2

Attaches GMP to the 5’ end

OHOH

Figure 12.19

OHOH

Figure 12.19

Adds methyl groups to guanine & riboses

Capping – Step 3

OH O

CH3

5 3

5

5 3

Endonuclease cleavage occursabout 20 nucleotides downstreamfrom the AAUAAA sequence.

PolyA-polymerase addsadenine nucleotidesto the 3end.

Polyadenylation sequence

PolyA tail

AAAAAAAAAAAA....AAUAAA

AAUAAA

AAUAAA

Polyadenylation

G/U

Splicing – Intron Sequences

35

5 splice site 3 splice siteBranch site

IntronExon ExonPy12N PyAGGA/CGGU Pu AGUA UACUUAUCC

Exon n ……A G G U A A G U …Intron …Y N Y Y R A Y …....Y12 N C A G N ….. Exon n+1

64 73 100 100 62 68 84 63 80 80 87 75 100 95 65 100 100

Branch Point

Yeast consensus

Intron loops out and exons brought closer

together

Figure 12.22

Splicing of Pre-mRNAs

Splicing of Pre-mRNAThis is a splicosome

Figure 12.22

Intron will be degraded and the snRNPs used again

Splicing of Pre-mRNA

Spliceosomal Cycle

Association of snRNAs with Pre-mRNA

Spliceosomal Complex

U2 snRNA interaction with branch point shown by compensatory mutagenesis

Potential interaction based on sequence complementarity

U2 snRNAMutations in branch points prevent splicing

Compensatory mutations in U2 snRNA restore splicing

Alternative Splicing of Tropomyosin

* Alternative pA*

* Alternative pA*

Alternative Splicing Regulates Drosophila Sex Determination

Myostatin Mutations in Cattle, Dogs, & Mice

Belgian Blue

Whippet

Mouse

Myostatin Splice Site Mutation

AGTgtaaatAGTgtaagt

cat/gtaagt

Wildtype myostatin Mutant myostatin

Exon 1

Exon 1

Exon 1 Exon 1

Exon 1

Exon 1Exon 2

Exon 2

Exon 2 Exon 2

Exon 2

Exon 2