BIOL200 RNA Processing II and Transport

RNA Processing II & Nucleocytoplasmic Transport

• RNA Processing II and Nucleocytoplasmic Transport lecture
• Lecture is PL2/3
• Lecturer: Holly J
• Course: BIOL200
• Date: November 20th, 2024

Housekeeping

• No lecture on December 4th
• End-of-term instructor review
• Submit questions by December 3rd at noon
• Sessions will be recorded
• Tutorials are one week behind the lecture schedule

mRNA Processing

• Modifications at the 5' and 3' ends of pre-mRNA are crucial for stability and protection
• Three major co-transcriptional steps:
  • 5' capping
  • 3' cleavage and polyadenylation
  • RNA splicing

5' Capping

• Nascent mRNA emerges from the RNA exit channel (25bps)
• A methylguanalate cap is added by a capping enzyme that interacts with RNA Pol II CTD
• Protects the mRNA, facilitates nuclear export, allows recognition by translation initiation factors

3' Cleavage & Polyadenylation

• Poly(A) signal = AAUAAA, + G/U
• Polyadenylation factors (CPSF, CStF, CFI, CFII) recognize the signal
• 3' end is cleaved
• 3' end is recognized by Poly A Polymerase (PAP)
• Slow Phase: 12 A residues added to 3' end
• Rapid Phase: PolyA Binding Protein (PABP) adds around 200 A residues

mRNA Splicing

• U1 (intron splice site) and U2 (branch point) interact with mRNA
• U4, U5, U6 are recruited
• U1 & U4 exit (active spliceosome)
• Transesterification Reactions
  • OH at branch point attacks 5' phosphate at the 1st intron residue, forming a lariat
  • 3' end of exon attacks 5' end of following exon
• Release of mature mRNA, snRNAs, and intron
• Self-splicing introns: in some cases, introns have the capacity to self-splice without external snRNAs

mRNA Splicing Specificity

• Exon cross recognition complex
• U2AF: Important for splicing efficiency
• SR Proteins: Contain RRM domains and arginine/serine rich domains
• Cooperative binding of U1 to the 5' splice site and U2 to the branch point
• Binding to exonic splicing enhancers (ESEs)
• ESES: sequences within exons that promote splicing

Alternative Splicing

• The presence of multiple introns allows for multiple variations of a protein from a single gene (protein isoforms)
• Example: Sexual differentiation in Drosophila
  • Sxl protein is only present in female embryos (early development)
  • Binding site (intronic splicing silencer) blocks use of splice site
  • Later in development, the gene is active in males and females
  • Males:
    • Absence of early Sxl protein => sex-lethal mRNA is spliced to make mRNA with a stop codon => no functional Sxl protein
    • No functional transformer protein
  • Females:
    • Presence of early Sxl protein => exon 3 is skipped => functional Sxl protein => binding of Sxl to ISS skips exon 2 => functional transformer protein
• Dsx protein (double-sex gene)
  • Females: complex of tra, Rbp1 and tra2 direct splicing, cleavage, and alt polyadenylation => short, female-specific Dsx
  • Binding site = exotic splicing enhancer
  • Males: no tra => exon 4 is skipped => longer, male-specific Dsx

RNA Editing

• Sequence of mature mRNA sometimes differs from the DNA's coding regions sequence
• Adenosine to Inosine (A -> I) OR Cytosine to Uracil (C -> U)
• Ex: Editing of apoB pre-mRNA changes C->U at position 666 in intestinal cells
• Shorter apoB-48 has N-terminal domains associated with lipids

Nucleo-Cytoplasmic Transport

Nuclear Pore Complex

• Proteins synthesized in the cytoplasm need to exit the nucleus (and potentially re-enter)
• Nuclear Pore Complex (NPC): made up of nucleoporins
  • Nuclear basket
  • Aqueous pore
  • FG nucleoporins: extended regions (Phe-Gly repeats + hydrophilic regions) form permeability barrier
  • Molecules up to 60kDA freely diffuse; larger molecules need to be transported

Nuclear Import

• Proteins wanting to enter the nucleus require a nuclear localization signal (NLS)
• Free importin binds to NLS on cargo protein, forming a complex
• Complex interacts with FG repeats in NPC channel
• Importin interacts with Ran-GTP causes cargo protein release (decreased affinity)
• Importin-Ran-GTP complex diffuses back to cytoplasm
• Ran interacts with Ran-GAP, hydrolyzes GTP to GDP, importin is released
• Ran-GDP returns to nucleus, interacts with Ran-GEF, causing GDP to be exchanged for GTP

Nuclear Export

• Proteins wanting to exit the nucleus require a nuclear export signal (NES)
• Free exportin1 forms complex with Ran-GTP and cargo protein (increased affinity)
• Complex interacts with FG repeats in NPC channel
• In cytoplasm, complex encounters Ran-GAP, stimulating GTP hydrolysis, cargo is released
• Exportin1 and Ran-GDP transported back to nucleus
• Ran-GEF stimulates conversion of Ran-GDP to GTP

mRNP Export

• Once mRNA processing is complete, it remains associated with proteins (mRNP complex)
• Exporter binds RNA with mRNP proteins during elongation (prior to processing)
• Forms a domain that interacts with FG repeats, allowing diffusion across NPC

Questions?