chapt 11

• The experimental tools to study RNA structure and function are more recently developed than the tools to study proteins and protein-encoding genes
• The human genome has about 22,000 protein-encoding genes
• In contrast, other genes are transcribed into non-coding RNAs (ncRNAs), RNA molecules that do not encode polypeptides
• In most cell types, ncRNAs are more abundant than mRNAs
• In a typical human cell, only 20% of transcription involves the production of mRNAs
• In part due to available experimental tools, a historical bias has existed against RNA; most biology instruction focuses on DNA and protein functions
• This chapter works to lessen that bias by exploring many examples of ncRNA structure and function as well as their role in plant and animal health
• ncRNAs


  Can bind to DNA or another RNA through complimentary base pairing, allowing them to affect the processes of DNA replication, transcription, and translation
• ncRNAs


  Can also bind proteins or small molecules, with stem-loop structures binding or scaffolding proteins or forming a binding site for a small molecule
• Common functions of ncRNAs

  • Scaffold
  • Guide
  • Alteration of protein function or stability
  • Ribozyme
  • Blocker
  • Decoy
• The key difference between a blocker and decoy is what they bind to; a blocker binds a molecule that is not an ncRNA and a decoy binds to an ncRNA
• Telomeres


  The ends of linear eukaryotic chromosomes composed of repeat sequences that protect the ends of the chromosomes from becoming tangled or broken
• As cells divide, the telomeres typically become shorter and eventually they are so short that the cell can no longer successfully divide; this causes the cell to go through a process of programmed cell death
• Some cells that divide rapidly express an enzyme called telomerase, which adds the repeating sequence and extends the telomeres
• Telomerase


  Contains both proteins and an ncRNA called TERC (telomerase RNA component)
• Telomere lengthening


  1. Binding of telomerase

  2. Polymerization (TERC acts as a template)

  3. Translocation (telomerase moves to the new end of the DNA strand and attaches another 6 nucleotides)
• HOTAIR


  An ncRNA in humans and other mammals that regulates transcription by forming a scaffold that binds 2 protein complexes and guides them to particular genes
• The protein complexes covalently modify histones, and these modifications silence the target genes
• The goal of the Fire and Mello experiment was to understand how the experimental injection of RNA was responsible for the silencing of particular mRNAs
• In the embryos that received single-stranded RNA, the mex-3 mRNA levels were decreased; in the embryos that received the double-stranded RNA, no mex-3 mRNA was detected - double-stranded RNA caused the mex-3 mRNA to be degraded
• RNA interference (RNAi)


  The phenomenon where double-stranded RNA causes the degradation of mRNA
• Sources of ncRNA that can promote RNA interference

  • MicroRNAs (miRNAs)
  • Small-interfering RNAs (siRNAs)
• miRNA synthesis and processing

  1. Transcription of primary-miRNA

  2. Cleavage of primary-miRNA to form precursor-miRNA

  3. Export of precursor-miRNA from nucleus

  4. Cleavage of precursor-miRNA by dicer enzyme to form 20-25 bp double-stranded miRNA
• siRNA and miRNA processing

  1. Precursor-miRNA and precursor-siRNAs are recognized by the dicer enzyme in the cytosol

  2. Dicer cuts the precursor molecules into 20 to 25 bp double-stranded miRNA or siRNA molecules
• RNA-induced silencing complex (RISC) formation


  1. Double-stranded miRNA/siRNA associates with proteins to form RISC

  2. One strand is degraded, the other is incorporated into RISC

  3. The single-stranded miRNA/siRNA acts as a guide for target mRNAs

  4. Once bound, the mRNA is inhibited from participating in translation or is degraded
• Primary-miRNA folding


  Hairpin (stem-loop) structure
• Primary-miRNA processing


  1. Cleaved to form precursor-miRNA

  2. Precursor-miRNA exported from nucleus
• Precursor-miRNA and precursor-siRNA processing

  1. Recognized by dicer enzyme in cytosol

  2. Dicer cuts into 20-25 bp double-stranded miRNA or siRNA molecules
• RNA Interference


  Mediated by microRNAs or small-interfering RNAs via the RNA-Induced Silencing Complex
• RNA-Induced Silencing Complex formation

  1. Double-stranded miRNA/siRNA associates with proteins

  2. One strand degraded, other incorporated into complex

  3. Single-stranded miRNA/siRNA complementary to target mRNAs

  4. miRNA/siRNA guides target mRNAs

  5. Bound mRNA inhibited from translation or degraded
• Signal Recognition Particle (SRP)

  • RNA-protein complex that targets polypeptides to the ER
  • In bacteria, composed of 1 ncRNA and 1 protein
  • In eukaryotes, composed of 1 ncRNA and 6 different proteins
• SRP RNA roles


  • Provides scaffold for protein binding
  • Stimulates GTPase activity of proteins in SRP and SRP receptor
• Protein targeting by SRP

  1. SRP binds to ER signal sequence, pausing translation

  2. SRP binds to SRP receptor in ER membrane

  3. GTP-binding proteins in SRP and SRP receptor hydrolyze GTP, releasing SRP and allowing translation to resume
• CRISPR-Cas system


  • Provides bacteria and archaea defense against bacteriophages and transposons
  • Type II CRISPR-Cas system provides defense against bacteriophages
• CRISPR-Cas type II system


  1. Contains CRISPR gene with repeats and spacers

  2. Contains tracrRNA gene

  3. Contains Cas genes
• CRISPR-Cas defense against bacteriophages

  1. Adaptation phase: Cas1/Cas2 complex inserts bacteriophage DNA fragment into CRISPR gene

  2. Expression phase: CRISPR, tracrRNA, and Cas9 genes expressed, tracrRNA guides crRNA to Cas9

  3. Interference phase: tracrRNA-crRNA-Cas9 complex binds and cleaves complementary bacteriophage DNA
• Abnormal expression of certain miRNAs associated with human cancers
• Some miRNAs are tumor-suppressors, others act as oncogenes
• HOTAIR highly expressed in several cancers, acts as an oncogene when overexpressed
• Various ncRNAs associated with neurological and cardiovascular diseases