rna splicing


Transcription of DNA into RNA, enzymatic reactions, RNA, RNA degradation

  • Transcription
    1. Initiation: promoter recognition, closed complex, open complex.
      • Promoter:
        • Prokaryotic: ←upstream, -35 region, Pribnow box, transcription start site (TSS, +1), downstream→
        • Eukaryotic: ←upstream, several upstream elements, TATA box, initiator element containing TSS (+1), downstream→
        • The high A-T composition in promoters facilitate unwinding of DNA.
        • Template strand = antisense strand = (-) strand = noncoding strand = the DNA strand that serves as the template for transcription.
        • Nontemplate strand = sense strand = (+) strand = coding strand = the DNA strand having the same sequence as the transcribed RNA.
      • Binding to promoter:
        • Prokaryotic:
          • holoenzyme = core enzyme (polymerase activity) + σ-subunit (promoter and strand specificity).
          • binding first forms the closed complex, and then DNA opens up, forms the open complex.
        • Eukaryotic:
          • A whole bunch of transcription factors (TFs) involved in promoter recognition, binding, and openning up DNA.
          • TBP = Tata binding protein. TAF = TBP associated factor.
          • Phosphorylation of Pol II C-terminal domain (CTD) opens DNA up, forms the open complex.
        • Polymerase must transcribe using the correct template strand. The σ-factor (prokaryotes) and TFs (eukaryotes) tell the RNA polymerase to bind the coding strand, while using the template strand as the template.
    2. Elongation:
      • Polymerases:
        • Prokaryotes have just one.
        • Eukaryotes have three:
          • 1. RNA Pol I: makes rRNA (except the small 5S rRNA that resembles a tRNA in size).
          • 2. RNA Pol II: makes mRNA.
          • 3. RNA Pol III: makes tRNA (and 5S rRNA).
      • Incorporation of NTPs.
      • Prokaryotes lose σ-subunit. Eukaryotes lose TFs.
      • Topoisomerases relaxing supercoils ahead and behind the polymerase.
      • Transcription-coupled repair: RNA Pol II encounters DNA damage, backs up, TFIIH comes along, recruits repair enzymes. Defective TFIIH → faulty transcription-coupled repair → Xeroderma pigmentosum and Cockayne syndrome (skin sensitive to sunlight radiation in both diseases).
    3. Termination
      • Prokaryotic:
        • Intrinsic termination: GC hairpin (stalls polymerase) followed by poly U (slips off).
        • Rho-dependent termination: ρ protein catches up to polymerase when it stalls at the hairpin, and bumps it off.
      • Eukaryotic:
        • Termination consensus sequence reached (AAUAAA).
        • Polymerase released somewhere further downstream to the consensus sequence.
  • RNA
    • 1. RNA = ribonucleic acid, has 2’-OH.
    • 2. rRNA = ribosomal RNA
      • Most abundant (r for rampant).
      • Catalyzes peptide bond formation in the ribosome.
    • 3. mRNA = messenger RNA
      • Longest (m for massive).
      • Contains sequence of codons for translation.
      • RNA splicing
        • pre-mRNA need to be processed.
        • Introns = interfering sequences, cut out.
        • Exons = spliced together.
        • RNA splicing proceeds via a lariat intermediate, by the action of the spliceosome (snRNPs), introns released in lariat form.
        • Some RNA can self splice.
    • 4. tRNA = transfer RNA
      • Smallest (t for tiny).
      • Contains anticodon.
      • Shuttles the correct amino acid to the correct codon during translation.
    • 5. snRNPs (snurps) = RNA + protein, involved in RNA splicing.
  • RNA degradation
    • RNases degrade RNA.
    • Post-transcriptional modifications protect RNA from degradation (5’ cap and polyA tail)
    • 2’-O-methylation prevents that position from attacking the RNA backbone.
Seeking SciNote, Biology: CRISPR


What do geneticists think will be possible when the the new gene-splicing CRISPR is fully operational on patients?


For those of us unfamiliar, CRISPR is a revolutionary new genetic splicing technology. Gene splicing refers to modifications to a gene transcript that can result in different proteins being made from a single gene. Interestingly, CRISPR’s inception began when dairy scientists discovered that bacteria used to create yogurt (by transforming lactose into lactic acid) had incorporated snippets of benign viruses into its genome. To their surprise, the incorporated DNA would create toxic agents to thwart infective viruses. In 2007, dairy scientists realized that they could effectively fortify bacteria by adding spacer DNA, which does not code for any protein sequence, from a virus. Then, five years later, as Time Magazine writer Alice Park skilfully describes, professors Jennifer Doudna and Emanuelle Charpentier noticed “up to 40% of bacteria developed a particular genetic pattern in their genomes. What they found were sequences of genes immediately followed by the same sequence in reverse, known as palindromic sequences. Further, bits of random DNA bases cropped up after each such pairing and right before the next one. After the dairy bacteria transcribed its spacer DNA and palindromic sequence into RNA, it self-spliced those segments into shorter fragments, with an enzyme called CAS9”. As you may be wondering, CRISPR stands for “clustered regularly interspaced short palindromic repeats”.

It is important for us to emphasize the versatility of this method. In the 2007 article, Doudna and Charpentier go into depth regarding the many benefits of the new genetic technology. These include the potential to “systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders”. As you might imagine, this opens up possibilities that were previously science fiction. Currently, painful blood transfusions are commonplace in the treatment of many diseases such as sickle cell anemia. Sickle cell affects red blood cells, which are made by stem cells in bone marrow. Soon, Massachusetts Institute of Technology synthetic biologist Feng Zhang envisions that this will soon no longer be necessary. She predicts that after doctors extract some of the marrow, scientists will splice out the defective fragment of DNA using CRISPR from the removed stem cells, then bathe the cells in a solution containing the non-sickle-cell sequence. As the DNA repairs itself naturally, it picks up the correct sequence and incorporates it into the stem cell genomes. After this one-time procedure, the stem cells would give rise to more red blood cells with the healthy gene. Eventually, the blood system would be repopulated with normal cells.

The treatment of HIV using CRISPR would be very similar. In this potential treatment, “patients would provide a sample of blood stem cells from their bone marrow, which would be treated with CRISPR to remove the CCR5 gene, and these cells would be transplanted back to the patient. Since the bone marrow stem cells populate the entire blood and immune system, the patient would eventually have blood cells that were protected, or “immunized,” against HIV”.

Despite this extraordinary potential, no biological technology comes without serious ethical concerns. As Jennifer Douda says herself, CRISPR “really requires us to careful thought to how we employ such a tool: What are we trying to do with it, what are the appropriate applications, how can we use it safely?”

Check out her book The Stem Cell Hope for learning about the future of stem cell technology.

Park, Alice. “A New Gene-Splicing Technique.” 100 New Scientific Discoveries: Fascinating, Unbelievable and Mind-expanding Stories. New York, NY: TIME, 2014. 92-95. Print.

Park, Alice. “It May Be Possible To Prevent HIV Even Without a Vaccine.” Time. Time, 6 Nov. 2014. Web.

Doudna, Jennifer A., and Charpentier, Emmanuelle (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096–1258096. doi:10.1126/science.1258096

Answered by: Teodora S., Expert Leader and Expert John M.

Edited by: Carrie K.