Post-transcriptional gene control

### Pre-mRNA Processing

• Occurs in the nucleus
• Includes 5' capping, 3' polyadenylation, intron removal & exon splicing
• Pre-mRNA transcripts are synthesized by RNA polymerase II

hnRNP

• Pre-mRNA is always associated with proteins
• hnRNP is a complex of hnRNA and proteins
• hnRNP proteins help prevent pre-mRNA folding to ensure proper processing and transport
• hnRNP also assists in transporting mRNA out of the nucleus

5’ Capping

• Occurs shortly after transcription initiation
• A 7-methylguanylate cap is added to the 5' end of pre-mRNA
• Capping enzyme associates with the CTD of RNA polymerase II
• Protects the 5' end from degradation and assists with export to the cytosol

Polyadenylation

• A multi-protein complex forms at the poly(A) signal in pre-mRNA
• The pre-mRNA is cleaved at a site generating a free 3' OH
• Poly(A) polymerase adds A residues to the 3’end
• This protects the mRNA from degradation in the cytoplasm

Intron Removal & Exon Splicing

• Introns are removed and exons are spliced together at every exon/intron junction
• Splicing requires conserved sequences at each end of every intron
• Invariant nucleotides include G-U at the 5’ splice site, A at the branch point, and A-G at the 3’ splice site

RNA Splicing Mechanism

• Splicing is carried out by a large ribonucleoprotein complex called the spliceosome
• The spliceosome contains snRNPs which are made of snRNA associated with proteins
• U1 snRNA binds to the 5’ splice site, U2 to the branch point, and U4/U6/U5 associate
• Two transesterification reactions lead to the removal of the lariat intron and ligation of exons
• The lariat intron is converted to linear RNA and degraded by nuclear exonucleases

Trans-Splicing

• Involves splicing together exons of separate RNA molecules, forming a single mature mRNA
• Occurs less commonly than cis-splicing
• Found in organisms like C. elegans, Trypanosomes, and Euglenoids

Alternative Splicing

• Different combinations of exons from the same pre-mRNA can be spliced together
• Results in different mature mRNAs (isoforms)
• Regulated by RNA-binding proteins

RNA Editing

• Exon nucleotides are altered in the nucleus prior to mRNA maturation
• The sequence of the mature mRNA differs from the genomic DNA
• Occurs rarely in higher eukaryotes, but common in mitochondria of plants and protozoans

Ribozymes

• RNA molecules with catalytic activity
• 23S and 28S rRNA have peptidyl transferase activity
• Group I and II self-splicing introns are found in various organisms

Nuclear Transport

• The nucleus is surrounded by two membranes with nuclear pore complexes (NPCs) for transport
• NPCs allow passive diffusion of small molecules and active transport of larger molecules
• FG nucleoporins form a sieve that selectively allows for macromolecule transport

Import into the Nucleus

• Proteins meant for use in the nucleus are synthesized in the cytosol
• These proteins contain a nuclear localization signal (NLS)
• Importins bind to the NLS and transport the cargo into the nucleus
• Ran-GTP mediates the release of the cargo from the importin in the nucleoplasm

Export out of the Nucleus

• Exportin transports cargo proteins containing nuclear-export signals (NES) out of the nucleus
• NES cargo binds to exportin and Ran-GTP
• Complex diffuses through the NPC
• Hydrolysis of Ran-GTP causes dissociation of the complex and exportin recycling

mRNP Transport out of Nucleus

• mRNA exporter protein directs most mRNPs through the nuclear pores
• This protein interacts with FG-nucleoporins as it progresses through the pore

GTP Switch Proteins

• Guanine nucleotide-binding proteins switch between active (GTP-bound) and inactive (GDP-bound) states
• GEF promotes GTP binding and GAP accelerates GTP hydrolysis