Module 6: Structure and function of RNA

• ribonucleic acid (RNA) is the product if transcription from DNA template (10x more RNA than DNA in a cell)
• Chemical structure of RNA:

  *ribose is used in place of deoxyribose

  -->ribose contains 2'-OH whereas deoxyribose lacks an oxygen

  -->this makes RNA less stable than DNA

  *the base Uracil is used in place of Thymine

  -->uracil doesn't have a -CH3
• Biological Roles of RNA:

  1. transfer of information from DNA to protein via mRNA
  2. synthesis of proteins (rRNA and tRNA)
  3. processing of messenger RNA (small nuclear RNA- snRNA- involved in splicing introns from pre-messenger RNA)
  4. processing and modification of ribosomal RNA
  5. catalytic RNA
• RNA content of cell:

  *a single cell contains 10pg (10^-11 g) RNA

  *80- 85% ribosomal RNA

  *10 -15% low m/w species (tRNAs, small nuclear RNAs)

  *1- 5% mRNAs
• Physical properties of RNA:

  *RNA is generally single stranded

  *RNA can base-pair with itself to fold into complex structures
• Regulation of usage of RNAs is controlled by:

  1. synthesis- transcription can be turned on and off rapidly (lac operon)
  2. regulates degradation (transferrin receptor mRNA)
  3. Translation efficiency- proteins can control the usage of certain RNAs (ferritin mRNA)
• RNA processing function summary:

  1. increase stability and translational efficiency
  2. splicing requires catalytic RNA
  3. splicing and editing increase the number of possible products arising from one gene
• mRNA capping

  *5' to 5' via the phosphates is not normal

  *5' phosphate- 3'-OH link

  *Methylation at 2'-position of first 2 nucleotides and on the added G

  -->linked to transcription initiation

  -->increases stability of mRNA

  -->required for efficient splicing

  -->nuclear export and translation initiation
• 3' Cleavage and Polyadenylation

  *G/U or U rich regions = signals of cleavage and polyadenylation

  1. cleavage and polyadenylation specificity factor (CPSF) binds to AAAAAA
  2. cleavage stimulatory factor (CstF) binds to G/U- recruits cleaving factors and poly-A-polymerase
  3. Cleavage and addition of PolyA

  *PolyA tailing functionally linked to transcription
• Pre-mRNA splicing- anatomy of an intron

  *Signals for splicing = conserved sequences at the 5' splice site, 3' splice site and branch point region

  *recognised by factors which remove introns
• Splicing

  1. cleavage at 5' splice site and lariat formation at branch point sequence
  2. cleavage at 3' splice site- removal of intron region and exon ligation
• Spliceosome assembly pathway

  *splicing carried out by small nuclear ribonucleoprotein particles (snRNPs) and protein splicing factors

  -->snRNPs = small nuclear RNA (snRNP) + numerous proteins

  *snRNPs involved in splicing are U1, u2, U4, U5 and U6

  *U2, U5 and U6 = the active site of the spliceosome

  U1 splice donor --> +U2 branch point --> +U4/5/6 displace U1 --> rearrange U2/5/6 - 'active site'
• Alternative splicing

  *exon inclusion/ exclusion- proteins with altered sequence or truncated if included exon has a stop codon

  *different proteins (isoforms) can be produced from a single gene