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CHAPTER 31 REGULATORY RNA

30.4 BACTERIA CONTAIN REGULATOR RNA
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Small untranslated anti-sense RNA species can regulate
gene expression in both bacteria & mammalian cells.
Control occurs in many levels: transcription termination,
RNA processing & degradation, translation initiation.
sRNA often encoded at the same loci as target RNA;
regulate by base pairing at the regions of
complementarity.
Bacterial regulator RNAs are called sRNAs.
Several sRNAs are bound by protein Hfq which increases
their effectiveness.
Tandem repeats can be transcribed into powerful
antiviral RNAs (immunity)
OxyS sRNA in E.coli:
a) OxyS = abundant, 109 nt, untranslated RNA induced
by oxidative stress
b) OxyS as pleiotropic regulator: regulate expression of
multiple genes  activates/repress expression of ~40
loci at post-transcriptional level
c) OxyS as anti-mutator, protect cells from spontaneous
and chemically induced mutagenesis.
OxyS inhibits expression of flhA (encodes for
transcription activator) by base pairing w/ a sequence
upstream of AUG initiation codon = Shine-dalgarno
sequence; inhibit ribosomal binding.

OxyS expression is regulated/repressed by OxyR at the
absence of oxidative stress. (H2O2)

10. CRISPR = cluster of regularly interspersed short
palindromic repeats; transcribed & processed into short
RNAs that function in RNA interference
a) CRISPR locus is transcribed into a larger RNA
precursor

11. CRISPR-Cas9 from Strep. pyogenes tool for genome
editing
a) Genome editing existing tools:
1) ZFN (zinc finger nucleases) & TALENS
(transcription-activator like effector nuclease)
- Generate mutations via double stranded
breaks to activate repair pathways in vivo
2) Homologous recombination of DNA
3) RNA interference (Limitation: temporary
inhibition of gene function & unpredictable offtarget effects)
b) CRISPR-Cas9 = Naturally existing adaptive immune
defense system in bacteria & archaea
- Acquisition phase: foreign DNA (carried by
phage) is incorporated into bacterial genome at
the CRISPR locus
- crRNA biogenesis: CRISPR locus is then
transcribed & processed into crRNAs (CRSPRRNA)
- Cas9 binding and RNaseIII cleavage to generate
mature crRNA/tra-crRNA/Cas9 complex
- Interference: CRISPR-Cas9 cleaves foreign
DNA containing a 20-nucleotide crRNA
complementary sequence (spacer) adjacent to
PAM sequence (NGG)

Efficient reproductive system: Maintained as hermaphrodites (reproductive organs associated w/ both male & female) . CRISPR nuclease system consists of 3 components: Cas9.Male exist for genetic studies . Induced double strand break leads to DNA repair via a) Homology directed repair (HDR) in the presence of donor template w/ homology to the targeted locus  precise gene editing/repair b) Non-homologous end joining (NHEJ). vary w/ small crRNAs a) Involve in sequence recognition & ds-DNA cleavage b) PAM = protospacer-adjacent motif (NGG) 2. Cleavage events & cell lineage development 3. male (XO. cheap to maintain) b) Short generation time: 3 days from egg to adulthood c) Small: 1mm . absence of donor template  insertions/deletions (indels). 959).Small genome size: 100Mb f) Transparency: development analysis from a single cells and all cell to be lineage 12.5 C. Discoveries of genetic regulation of organ development & programmed cell death (to maintain fixed no. of cells) 14. feed on nonhazardous bacteria. Cas9 = CRISPR associated protein.ELEGANS AS MODEL SYSTEM 1. of cells: female (XX. 1031) e) Amenable for genetic analysis: . elegans: a) Easily cultivated (grow on petri dish. contain 2 nuclease domains. crRNA & tra-crRNA a) Guide RNA (sgRNA) = crRNA & tra-crRNA 13. Features of C. disrupt target locus 30.d) Few/fixed no.

complexed w/ RISC proteins . siRNA guide RISC to target mRNA  RISC degrades target mRNA Dicer = an endonuclease that process double-stranded precursor RNA to 21-23 nucleotides RNA interference molecules Functional domains in dicer include: helicase.6 RNA INTERFERENCE BY SYNTHETIC SMALL INTERFERING DSRNA (SIRNA) 1. RNA interference by in vivo transcribed siRNA a) Transfection of shRNA construct (shRNA = short hairpin RNA) Distance between PAZ & RNaseIII determine the length of siRNA product. 3.4. DUF283 (dsRNA binding domain). 5. elegans. RNA-interference mechanism: Initiation: Dicer cleaves long dsRNA to ~21bp small interfering RNA w/ 2-nucleotide 3’ overhangs (more effective than long dsRNA) 30. responsible for RNA interference (= gene silencing by double-stranded RNA) Experiment findings: a) Injection of anti-sense RNA to silence a gene in C. Transfection of long dsRNA (>30nt) into mammalian cells causes non-specific suppression of gene expression. elegans to assess gene function  work b) Injection of dsRNA  more potent effect Effector: formation of RISC (RNA-induced silencing complex). 2. shRNA duplex w/ loop structure. RNase III. PAZ (recognize & bind 2-nt 3’ overhang) 4. Discovery of small interfering RNA (siRNA) in C.

epidermis. miRNA within a genomic cluster are often related to each other. Phenotype of lin-4. One miRNA can inhibit over 100 different target genes. formation of organs) require proper timing b) Heterochrony = change in the relative timing of developmental events. 3.Affect different developmental events: cell division pattern. poorly formed adult 10. animal & fungi (eukaryotes) miRNA originate from pri-miRNA = 5’-capped & 3’polyA full length precursors synthesized by RNA pol II. let-7 (stRNA) were identified in C. 5.elegans a) Each stage of development (cycle of cell divisions. lin-14 mutants: a) Adult lin-14 LOF mutant: lack many adult structure.Retarded: developmental events are repeated d) Heterochronic mutation: .Affect different tissues: intestine.elegans (Founding members of an abundant class of tiny RNAs world) Post-transcriptional regulation of heterochronic gene lin14 by lin-4 mediates temporal pattern formation in C. neurons . Regulatory pathway between lin-4 & lin-14 11. Different types of miRNA: a) stRNA (short temporal RNA): modulates mRNA expression during development b) piRNA: regulate gene expression in germ cells and act to silence transposable elements c) siRNA: complementary to virus & transposable elements First miRNA lin-4. 2 general phenotypes seen in heterochronic mutants: . 2. muscle. Micro-RNA (miRNA) are endogenous ~22nt RNA regulate gene expression in plant. 7. growth of tissue.c) 8. 6. 4. differentiation Effects of lin-4. cell cycle length. 9. let-7 miRNA: .Precocious: developmental events are skipped . 7. Structure & sequence of lin-4.7 MICRO-RNA ARE WIDESPREAD REGULATORS IN EUKARYOTES 1. lin-14 is required for the timing of cell division in L1 stage (lin-14 LOF  L1 is skipped) b) lin-4 regulates transition from L1 to L2 (lin-4 LOF  L1 is repeated) Application of siRNA in biomedical research: Useful & effective tool for specific gene knockdown Limitations: off-target effect Minimization of off-target effects: a) Careful design of shRNA construct b) Introduce 2 or more siRNA c) Rescue undesired phenotype caused by an siRNA w/ an siRNA resistant expression construct 30. 6. lin-14 mutation on the timing of developmental events: a) 5. unable to lay eggs due to failure in vulva development  accumulation of eggs b) lin-4 LOF mutant: develop certain adult feature precociously at larval stage  small.

let-7 controls L4-to-adult transition in C. all involve in miRNA mediated translation repression. 6. only Ago-2 involve in mRNA cleavage) lin-4. 5. let-7 is conserved across several species. 5’end w/ less bp will be chosen preferentially c) Passenger RNA: other strand of mature miRNA that is degraded Generation of RISC . Small RNAs are produced by processing of longer precursors. b) Guide RNA: a strand in mature miRNA that is incorporated into RISC. let-7 mutant: seam cells fail to terminally differentiate and continue to divide  bursting through vulva 14. let-7 RNA precursor form a hairpin structure. a) Base pairing of miRNAs (5’-3’) to the 3’ UTR of target mRNAs (lin-14. leave a 2-nucleotide overhang at 3’ end c) Pre-miRNA after Drosha cleavage is 65-70nt long Dicer: convert pre-miRNA to mature miRNA a) Occur in cytoplasm. miRNA regulation of gene expression is not exclusive to C. mature miRNA is shown as blue (lin-4) & red (let-7) 12. miRNA is higher eukaryotes 30.elegans 4. Action of lin-4 & let-7: 3. (primary) pri-miRNA  pre-miRNA  miRNA duplex 2. Drosha & dicer are RNase III (endonuclease). elegans. mature miRNA is generated in cytoplasm b) Cleavage also leaves 2-nucleotide overhang at 3’ end c) miRNA after dicer cleavage is 21-23nt long RISC (RNA-induced silencing complex) = a ribonucleoprotein particle composed of a short singlestranded siRNA and a nuclease that cleaves mRNAs complementary to siRNA (receive siRNA from dicer and delivers it to mRNA) Components in RISC: a) Argonauts protein (4 in mammals. lin-14 is one target. both process double-stranded long RNA transcripts Drosha: convert pri-miRNA to pre-miRNA a) Occurs in nucleus b) Cleavage sites are 11bp away from dsRNA & ssRNA junctions.1. lin-41) b) Binding site between lin-41 & let-7 maybe recognized by RBP (RNA binding proteins) 13.8 PROCESSING OF MIRNA IN EUKARYOTES 7.

it can also silence host genes 30. 3.8 RNA INTERFERENCE MODE OF ACTION 1. miRNA regulate gene expression via base pairing w/ complementary sequences in target mRNA RNA interference trigger degradation.9 HETEROCHROMATIN FORMATION REQUIRES MIRNA . 4. translation inhibition or mRNA activation. Inhibition of mRNA circularization via deadenylation Promote deadenylation & decapping Double-stranded RNA (injected from external source): inhibit translation. miRNA mode of actions in different species: a) Plants: mRNA degradation b) Animals: translational regulation c) Plant & animals. 2.e) f) 5. trigger mRNA degradation in mammalian cells (sequence specific effects). yeast: transcriptional regulation (histone methylation to inactivate chromatin) Mechanisms of miRNA mediated gene silencing: a) Inhibition of translation elongation b) Co-translational protein degradation c) Competition for the cap structure d) Inhibition of ribosomal subunit joining 30.