Post-Transcriptional Processing

• 18S, 5.8S and 28S rRNA are transcribed by RNA Polymerase I as a single 45S precursor
• Processing of rRNA occurs in the nucleolus
• 5S rRNA is transcribed separately by RNA polymerase III
• Modifications of nucleotides confer additional fitness to assembled ribosomes, allowing the subunits to come together more efficiently and enabling more efficient translation
• modification of nucleotides during post-transcriptional processing of rRNA involves the addition of methyl groups
•  Processing rRNA

  1. Nucleotides are modified
  2. Pre-rRNA is assembled with ribosomal proteins
  3. Pre-rRNA is cleaved into 18S, 28S and 5.8S
• tRNA is transcribed by RNA polymerase III
• Processing of tRNA 

  1. Nucleotides are cleaved from the ends
  2. Nucleotides CCA are added to the 3' end
  3. Some nucleotides are modified
  4. Intron is removed and products are ligated
• Cleavage at the 5' and 3' ends by nucleases occurs during the processing of tRNA
• Addition of CCA to the 3' end of tRNA provides an important recognition site for attachment of the correct amino acid
• Nucleotides around the anticodon of tRNA are particularly modified
• mRNA is the most processed RNA
• In prokaryotes, transcription occur in the same place and at the same time
• In eukaryotes, pre-mRNA transcripts are extensively processed in the nucleus and transported into the cytoplasm before translation can occur
• Capping at the 5' end occurs when the transcript is not fully complete
• Capping adds a methylated guanine to the transcript through a 5'-5' linkage with GTP
• Capping is done by three enzymatic interactions
• Capping protects the 5' end of mRNA from phosphatases and nucleases
• Capping also enhances mRNA translation by providing a docking site
• Addition of a 3' poly(A) tail occurs after transcription has ended
• Pre-mRNA is cleaved and roughly 250 adenylate residues are added using ATP as the substrate
• The poly(A) tail is not encoded by DNA
• The poly(A) tail increases mRNA stability and enhances translation
• Deadenylation is associated with mRNA decay
• All eukaryotic mRNAs are polyadenylated except for histone mRNAs
• Histone mRNAs have a stem-loop structure followed by a purine rich sequence to direct cleavage
• The purine rich sequence of histone mRNA attracts a complex with the complementary sequence, recruiting endonucleases to cleave the mRNA
• Oligo-dTs bind to the poly(A) tail, which are bound to magnets to allow mRNA to be isolated by applying a magnetic field
• Processing mRNA

  1. Capping at the 5' end
  2. Addition of the 3' poly(A) tail
  3. splicing
• Exons = coding regions
• introns = non-coding regions
• Introns can be very long
• Splicing removes the introns and links the exons to form mature mRNA
• Consensus sequences at the ends of introns identify splice sites
• The branch site is located upstream of the 3' splice site
• Mutations in splice sites or branch sites leads to abnormal splicing, which can shift the frame and produce a defective protein
• The spliceosome is a large splicing complex consisting of snRNAs, proteins and the pre-mRNA to be spliced
• Mutations can occur in the pre-mRNA or in the splicing factors
• Point mutations can incorrectly code for a splice site, leading to incorrect splicing
• Alternative splicing is removing a specific exon to generate diversity