MCDB 30H

• Spliceosome components


  • U4
  • U6
  • U5
  • U1
  • U2
• Spliceosome


  • Highly conserved (yeast to metazoans)
  • 5 snRNAs
  • Over 100 proteins
• The splicing reaction


  1. 1st step

  2. 2nd step
• The splicing reaction is a two step reaction that forms a lariat and requires energy (ATP)
• DExH/D proteins


  Bind and hydrolyze ATP (energy) to help drive rearrangements at every step of splicing
• Spliceosome rearrangements are ATP dependent
• Prp16 uses ATP energy


  To drive rearrangements
• Yeast genetic screens can be used to answer questions about how DExD/H-box proteins use energy to drive rearrangements
• Prp5


  ATPase activity removes Cus2 and remodels U2 snRNA
• Deletion of Cus2 suppresses Prp5 ATPase mutations, and U2 mutants suppress Prp5 ATPase mutations
• Transposon screen to identify effectors of DEAD-box protein helicase activity


  1. Transform transposon library into mutant strain

  2. Screen for suppressors of growth defects

  3. Identify mutants by PCR
• The name of the sequences that interrupt genes are transposons
• Lab overview

  1. TRANSFORM cells with mutagenized yeast DNA

  2. Spread cells onto plates that allow "selection" of library mutations

  3. Make "replicas" of this plate
• Prp16-2


  Yeast DNA
• Temperature is 25°
• Spread cells onto plates


  Allows "selection" of library mutations
• Replica plating


  Make "replicas" of this plate
• Temperature is 36°
• DNA


  Deoxyribonucleic acid
• RNA


  Ribonucleic acid
• Transcription


  The process of copying genetic information from DNA to RNA
• Protein


  Large biomolecule composed of one or more long chains of amino acid residues
• Translation


  The process of synthesizing a protein from the information encoded in mRNA
• Gene


  The entire nucleic acid sequence that is necessary for the synthesis of a functional gene product (polypeptide or RNA)

  
• Pre-mRNA


  The initial RNA transcript before splicing
• A gene is more than the nucleotides encoding the amino acid sequence in the protein (i.e. the coding region)
• Intron


  Non-coding sequence within a gene that is removed by RNA splicing
• Exon


  Coding sequence within a gene that is retained after RNA splicing
• A gene includes regions involved in transcriptional regulation (e.g. promoter, transcriptional enhancers)

  
• A gene includes a sequence that defines the 3' end of the synthesized RNA (i.e. the Polyadenylation signal)

  
• DNA is wound around proteins for compaction
• A gene includes non-coding regions that interrupt it (i.e. introns)
• Nearly all mammalian genes contain introns
• To express a gene

  Noncoding sequences are removed, coding sequences ligated together

  
• Introns make up the majority of the gene sequence, being 25x the length of exons
• Exon


  Segment of a gene that reaches cytoplasm as part of the mature mRNA

  
• On average there are 10-12 introns per gene, but up to 100
• RNA splicing is a really important reaction
• Intron


  Segment of a gene that is removed by splicing of the pre-mRNA and is not included in the mature mRNA or exported

  
• mRNA splicing is a dynamic process involving over 100 proteins and 5 small nuclear RNAs