GTS

• Post-transcriptional processes


  • Capping of pre-mRNA
  • Polyadenylation of pre-mRNA
  • Splicing of pre-mRNA

  
• Post-transcriptional processes do not necessarily follow in a step-wise manner, often occur simultaneously

  
• One post-transcriptional process is coupled to another
• Post-transcriptional processes are also coupled to transcription itself

  
• mRNA capping


  1. Methylated guanine is added to the 5' end

  2. Unique 5'-5' covalent bond used

  3. Three phosphates separate Me-G from the first mRNA residue

  
• Enzymes involved in mRNA capping

  • Guanylyl transferase: adds G to 5' end
  • Guanine methyl-transferase: adds Me to N7 of G

  
• Only mRNA is capped and translated, not other RNA species
• Structure of 5' methylated cap

  Methylated guanine with 3 phosphates separating it from the first mRNA residue

  
• Capping initiates and enhances translation of mRNA by the ribosome


  • Capping occurs only in eukaryotes
  • Translation requires 40S ribosomal subunit to bind to Me-G cap
  • Eukaryotic mRNAs are typically monocistronic
  • Translation initiation factor eIF4E recognizes the 5' Me-cap

  
• Kozak's rules


  • AUG triplet must be set within a consensus sequence GCCA/GCCAUGG or ACCAUGG
  • Ribosome moves along mRNA looking for this sequence to initiate translation

  
• Capping protects mRNA and facilitates transport

  • Uncapped mRNA have a free 5'-phosphate group that can be degraded by RNases
  • Cap-binding complex (CBC) recognizes and binds to Me-G cap, facilitating transport from nucleus to cytoplasm

  
• Capping regulates mRNA elongation

  • RNA polymerase pauses after a few nucleotides are made, 5' cap is added during this pause
  • Pausing is released when pTEF-b kinase is recruited, causing phosphorylation of RNA polymerase II

  
• Polyadenylation of pre-mRNA


  1. Cleavage of transcript at polyA site

  2. Addition of polyA tail

  
• Polyadenylation has various roles

  • Increases stability of mRNA by protecting 3' end from degradation
  • Regulates efficiency of translation
  • Functions in regulation of controlled mRNA degradation

  
• Standard histone mRNAs are not polyadenylated, are protected from degradation by a stem-loop structure at 3'-end
• Polyadenylation is linked to transcription, translation, and mRNA degradation

  
• RNA splicing


  Removes intervening sequences (introns) and joins exons together
• RNA splicing occurs in the spliceosome

  • Consists of 5 different U RNAs and over 100 splicing factor proteins
  • Spliceosome assembly is ordered, forms a large and small subunit
  • Tunnel in spliceosome accommodates the pre-mRNA to be spliced

  
• Consensus sequences around 5' and 3' splice sites

  • Almost all eukaryotic introns begin with GU and end with AG
  • Pyrimidine-rich region (polypyrimidine tract) is located upstream of 3' AG acceptor splice site
  • A residue upstream of pyrimidine tract is the branch point

  
• Splicing proceeds in 2 transesterfication reactions

  1. Ester bond between 5' phosphate of intron and 3' oxygen of upstream exon is exchanged for ester bond between 5' phosphate of intron and 2' oxygen of branch point

  2. Ester between 5' phosphate of downstream exon and 3' oxygen of upstream exon joins upstream and downstream exons, releasing intron as a lariat

  
• Role of snRNPs in splicing

  • U1 snRNP binds to 5' donor site
  • U2 snRNP binds to branch point
  • U5 snRNP binds to upstream exon, followed by U4/U6 snRNP at 5' splice site
  • U1 snRNP and U4 RNA is released, U6 snRNP releases U1
  • U6 and U2 particles interact, binding 5' splice site close to branch point
  • Separation of upstream exon from intron, lariat formation, and joining of upstream and downstream exons

  
• How U-RNAs recognize the mRNA molecule

  • Normal Watson-Crick base pairing
  • U1 RNA binds to 5' splice site, U2 snRNP recognizes branch point region

  
• Why upstream exon does not float away after cleavage

  Spliceosome component hSLu7 holds it in close proximity to the AG of the correct 3' splice site
• Role of SR proteins in splicing

  • Mediate recruitment of snRNPs to pre-mRNA
  • Determine which splice sites will be joined
  • Bind to exon-splicing enhancer sequences (ESEs) within exons to ensure correct exons are included

  
• Role of SR proteins in exon skipping and alternative splicing

  • Exon skipping: If mutation occurs in ESE that prevents SR binding, affected exon will be incorrectly spliced out
  • Alternative splicing: Normal process where exons are spliced together in different combinations to generate different mature mRNAs

  
• Transcription and RNA processing in the nucleus are coupled

  • As RNA polymerase transcribes, it becomes associated with proteins that mediate capping, splicing, and polyadenylation
  • Recruitment of these factors is linked to phosphorylation of the C-terminal domain of RNA polymerase II

  
• Coupling of transcription initiation, processing and elongation - role of RNA polymerase II

  • C-terminal domain (CTD) of RNA polymerase II plays a crucial role
  • Phosphorylation of serine 5 stimulates transcriptional initiation and recruitment of capping factors

  
• Coupling of transcription initiation and processing - role of the spliceosome

  • Spliceosome interacts with CTD of RNA polymerase II, linking capping and splicing
  • Spliceosome interacts with CPSF, linking splicing and polyadenylation

  
• Chain elongation by RNA polymerase is linked to RNA-processing complexes

  
• RNA splicing can take place co-transcriptionally or after transcription has completed


  • Introns may be removed while transcription is still occurring (co-transcriptional splicing)
  • Introns may be removed after transcription has completed (post-transcriptional splicing)

  
• U2 snRNP


  Can interact with CPSF to couple splicing and polyadenylation

  
• CTD of RNA polymerase II

  Composed of many repeats of a 7 amino acid sequence, allowing many proteins to associate with a single RNA polymerase II

  
• Presence of 5' capping, splicing and polyA factors


  Enhances rate and specificity of RNA processing when splice sites and PolyA signals are being transcribed

  
• Association of RNA splicing factors with Ph-CTD

  Stimulates transcription elongation

  
• RNA splicing


  Can take place co-transcriptionally or after transcription has completed

  
• Co-transcriptional splicing


  • Introns may be removed while transcription is still occurring, before polyadenylation

  
• Post-transcriptional splicing


  • Introns may be removed after transcription has completed, after capped-polyadenylated mRNA has been released from DNA

  
• Tight packing of nucleosomes


  Slow elongation of transcription, sufficient time for co-transcriptional splicing
• Open chromatin


  Rapid transcription elongation, not enough time for all introns to be removed, splicing continues after mRNA release

  
• Trimethylation of histone H3 at arginine position 4


  More open chromatin, enhances recruitment of FACT protein involved in elongation and U2 snRNP involved in splicing, therefore enhanced transcriptional elongation is linked to enhanced splicing