L13: RNA Splicing

• What are eukaryotic genes often split by?

  Introns
• What is the purpose of RNA splicing?

  To remove introns and join exons in the final mature RNA product
• What technique was used to discover split genes?

  R-loop analysis
• What does a single displaced loop of ssDNA indicate in bacterial R-loop analysis?

  That the gene is contiguous and has no introns
• Who independently analyzed adenoviral genes and mRNAs in 1977?

  Sir Richard J Roberts and Phillip A Sharp
• What did Sharp and Roberts discover about mRNA and DNA sequences?

  They found multiple loops indicating non-contiguous sequences of DNA
• What indicates the presence of an intron in eukaryotic R-loop analysis?

  Two displaced strands of ssDNA and a loop of dsDNA
• What is an intron?

  A nucleotide sequence within a gene that is removed by RNA splicing
• What is an exon?

  A nucleotide sequence that remains in the final mature RNA product after splicing
• How does intron possession vary among different organisms?

  Vertebrates have mostly split genes, while yeasts may lack introns
• What is the typical length of most exons?

  Less than ~1,000 nucleotides
• How do introns vary in size?

  They can vary from 50 to 20,000 nucleotides
• What was the conclusion from the Tetrahymena study regarding introns?

  The intron is excised from the primary transcript
• What did Cech discover about splicing in low salt conditions?

  There was minimal splicing
• What was the significance of adding GTP to transcripts in Cech's experiments?

  It stimulated splicing in vitro
• What is the role of the co-factor in the splicing mechanism?

  The co-factor attacks the phosphate at the 5’ splice site
• What is transesterification in the context of RNA splicing?

  It is the process of exchanging the organic group of an ester with an alcohol
• What is the take-home message regarding Tetrahymena rDNA introns?

  They can self-splice in the absence of any protein if guanosine or its derivatives are present
• What are the two classes of self-splicing introns?

  Group I and Group II introns
• What are the conserved features of introns?

  5’ splice site, 3’ splice site, and branch site
• How do Group II introns perform splicing?

  They fold and use the 2’-OH of the branch site adenosine to attack the 5’ splice site
• What structural feature aids Group II introns in splicing?

  The base-paired secondary structure that brings splice sites close together
• What splice site is conserved in all classes of introns?

  The 3’ splice site is conserved in all classes of introns.
• What is the role of the 2’-OH of the branch site adenosine in Group II introns?

  It attacks the phosphate at the 5’ splice site during splicing.
• What type of bond is formed when the adenosine in Group II introns has three phosphodiester bonds?

  One of the bonds is an unusual 2’, 5’ phosphodiester bond.
• What structural changes occur in Group II introns during splicing?

  The intron folds, bringing the 5’ and 3’ splice sites close together.
• What are the functions of Group II introns in splicing?

  • Can self-splice in vitro in high salt concentrations
  • Require splicing factors in vivo
  • Some encode maturases to improve splicing efficiency
  • Some encode homing endonucleases
• Where are Group I introns commonly found?

  In the nuclear genomes of protists, rRNA genes, and mitochondrial genes of animals and fungi.
• Where are Group II introns found?

  In rRNA, tRNA, and mRNA of mitochondria in fungi and protists.
• In which organisms have Group I introns been found sporadically?

  In bacteria, particularly in bacteriophages of Gram-positive bacteria.
• What does the intron-early hypothesis suggest about introns?

  It suggests that introns are of ancient origin and play a valuable role in modern organisms.
• What is a snRNP?

  A snRNP is a small nuclear ribonucleic particle involved in splicing.
• What are the steps involved in spliceosome-dependent splicing?

  1. U1 binds the 5’ splice site.
  2. U2 binds the branch site.
  3. A trimer of U4/6 and U5 binds.
  4. U1 covers the 5’ splice site and U4 inactivates U6.
  5. The spliceosome assembles and brings splice sites close together.
• How does the phosphorylated C-terminal domain of RNA Pol II relate to splicing?

  It recruits spliceosomes, coordinating intron removal with transcription.
• What advantages do nuclear introns provide?

  • Greater efficiency of removal
  • Nuclear control of splicing
  • Coordination of intron removal with transcription
  • Maintenance of introns in the genome for potential benefits
• What is alternative splicing and its significance?

  • Mechanism that generates protein diversity
  • Can be controlled developmentally
  • Example: calcitonin gene produces calcitonin and CGRP
• What is exon shuffling and its potential impact?

  • Recombination of exons during DNA breaks and rejoining
  • Can lead to novel proteins
  • Example: ERdj5 protein with domains from Hsp40 and PDI
• What can mutations in splice sites lead to?

  They can lead to genetic diseases due to errors in splicing.