Biochem Post-Transcriptional Modification (Eukaryotes ONLY)

Translation-Ready mRNA Contains:

• 5’ and 3’ untranslated regions (UTR)

• Open reading frame between the UTRs

• 5’ methyl cap

• 3’ poly A tail

C-Terminal Domain

• Contains a YSPTSPS repeat (an unstructured segment)

• Serine residues are phosphorylated by kinases

Why is phosphorylation of RNA Pol 2 CTD necessary?

• Promoter escape

• Serves as docking site for many RNA processing enzymes

  • capping enzyme

  • cap binding complex

  • cleavage/termination complexes

  • RNA splicing factors

5’ Cap

• 7-methylguanosine on 5’ end of mRNA

• Formed via a 5’,5’-triphosphate linkage

Methyl groups derived from

SAM (s-adenosylmethionine)

Cap-Synthesizing Complex

• Bound to Pol 2 CTD

• Adds 5’ Methyl Cap

Phosphohydrolase

Removes gamma phosphate in process of adding methyl cap

Guanylyltransferase

In addition of methyl cap it grabs a GMP from GTP

Functions of 5’ Methyl Cap

• Protects mRNA from RNases

• Binds to cap-binding complex to keep RNA attached

• Needed to interact with translational machinery

  • Participates in ribosome binding

Pol A Tail

• String of A residues added to 3’ end

• Helps protect RNA from enzymatic destruction

Polyadenylate (polyA) Polymerase

• Catalyzes addition of A residues to free 3’ OH group

• Template-independent RNA Pol

RNA Splicing

• Removal of introns

• Linking of exons

• Catalyzed by spliceosome

Spliceosome

• Enzyme complex that binds to introns to cut them out

• Contains small nuclear ribonuclear proteins (snRNPs)

  • RNA of these has enzymatic function

Lupus

• Autoimmune disease

• Individuals generate antibodies against protein portions of their spliceosome

• Abnormal proteins result

snRNA

• Small nuclear RNA

• Within snRNP

• Enzymatic portion

U1 snRNP

Binds to 5’ splice site in RNA

U2 snRNP

Binds to branch site and aligns mRNA for first splicing

U4 snRNP

Binds to and sequesters U6 snRNP

U5 snRNP

Aligns the pre-RNA for second splicing reaction

U6 snRNP

Promotes catalysis of splicing reactions

GU/AU Rule

• Defines splice sites

• All sequence in between and including GU at intron 5’ end and AG at intron 3’ end are removed

Splice Junction

Links the 5’ exon to the 3’ exon

Branch Point A

• Within the intron

• 2-OH acts as nucleophile for splicing reaction

U4 _____ U6 catalytic activity when bound.

blocks

Spliceosome Assembly (ATP Required)

1. U1 snRNP recruited to 5’ splice site

2. U2 recruited to branch site A

3. U5 and U4/U6 bind to complex

Activation of Spliceosome

1. U1 and U4 exit

2. U2, U5, U6 base pair to each other

   1. Aligns branch site with 5’ splice site for first transesterification

3. They rearrange for second transesterification

Bond Created During Splicing Reaction

2’-5’ Phosphodiester Bond

First Transesterification in RNA Splicing

2’-OH of branch point A attacks 5’-P

• Attacks the 5’ splice site of the intron

Second Transesterification in RNA Splicing

• 3’OH uf upstream exon attacks 5’ P of downstream exon (3’ splice site)

• Intron released in lariat structure (looks like lasso)

Hemophilia

• Splicing defect from error in selection of 3’ splice site

• Base change in second A to G

  • Splices at this AG instead of further down

• Frameshift Mutation

• Incorrect amino acids in translation result

Frameshift Mutation

Changes how the ribosome reads the open reading fram

Alternative Splicing

Process in which a particular exon may or may not be incorporated into the final mRNA

• Depends on RNA-binding splicing factors expressed in that cell

• Cell specific

CALCA Gene

• Mutually exclusive exons

• Calcitonin in thyroid

  • lowers blood calcium, tumor marker for cancer growth

• Calcitonin Gene-Related Peptide in neurons

  • Vasodilator

In alternative splicing, sequence-specific RNA-binding proteins…

• Tell the spliceosome which sites to use

• Bind to sequences in introns and exons of primary transcript

SR Proteins

• Serine/Arginine Rich

• Bind exonic splicing enhancers (ESE)

• Promote splicing

Heterogeneous Nuclear RIbonuclear Proteins (hnRNP)

• Bind to exonic splicing silencers (ESS)

• Inhibit splicing

Nuclear Export of mRNA

• Cap binding complex binds to nuclear pore complex

• mRNA thread 5’ to 3’

• Cytoplasmic proteins replace nuclear ones

RNA Interference (RNAi)

Group of mechanisms in eukaryotes involving small ncRNAs that usually reduce expression of specific genes

MicroRNA (miRNA)

Primary RNAi mechanism in mammals

Small Interfering RNA (siRNA)

Primary RNAi in plants

• promotes mRNA endonuclease cleavage

What transcribes miRNA?

RNA Pol 2

Endonuclease Drosha/DGCR8

Cuts out one hairpin of transcribed miRNA

• This is a secondary structure

Dicer

• Cytoplasmic endonuclease

• Removes loop of hairpin of miRNA

RNA-induced silencing Complex (RISC)

• mature miRNA is loaded into this protein complex

• binds to target mRNA promoting…

  • mRNA degradation

  • recruitment of RNases

  • translational repression

Hereditary Amyloid Transthyretin (hATTR)

• Progressive fatal disease

• Missense mutation

• Onpattro first FDA approved siRNA based drug to treat

  • Allows for degradation of mutant strand