RNA interference (RNAi)

  • RNAi has mainly two purposes:
    1. degrade hostile RNA from viruses and transposons (exogenous RNAi)
    2. regulate gene expression on a post-transcriptional level (endogenous RNAi)
  • Initially, double stranded miRNA/siRNA is cleaved into shorter fragments by the endoribonuclease enzyme Dicer.
  • Interaction complex that is formed by single stranded miRNA/siRNA, targeted mRNA and proteins is called “RNA induced silencing complex” (RISC).
  • The formation of the RISC will either result in degradation of target mRNA or in repression of translation of target mRNA.
    • mRNA is degraded if pairing of miRNA/siRNA is fully complementary.
    • Translation of mRNA is repressed/disturbed if pairing of miRNA is not fully complementary.

MicroRNA (miRNA)

miRNA biogenesis
miRNA biogenesis
  • mediates RNAi in an endogenous manner
  • 21 – 25 nucleotide small RNA
  • non-coding
  • binds to the 3′-UTR of target mRNA.
  • miRNA genes may be located within introns and exons of other (protein-coding or non-coding) genes.
    • In these cases, miRNA genes may be transcriptionally regulated through the promoters of these genes.
  • MicroRNA genes are first transcribed into primary miRNA (pri-miRNA), which is further processed to pre-miRNA.
    • pre-miRNA is exported to the cytoplasm, where it is cleaved by the Dicer enzyme into shorter RNA molecules called imperfect miRNA (miRNA*).
    • miRNA* is separated into single strand RNA molecules, from which usually one (guide strand) acts as the final mature miRNA, that interacts with the target mRNA.
  • If miRNA binds partial complementary, translation is inhibited.
  • If miRNA binds full complementary, target mRNA is degraded.
  • MicroRNA function in general seems to be linked to multicellularity:
    • Stem cells don’t need functional miRNAs for viability, but they fail to differentiate without miRNAs.
    • miRNAs are essential for vertebrate development.
    • miRNAs are involved in differentiation and maintenance of cell identity.
  • Examples
    • miR-143 and miR-145 expression is downregulated in various human cell lines derived from breast, prostate, cervical, lymphoid cancers, and, particularly, colorectal tumors.
    • Predicted targets for the miR-17-92 cluster include tumor suppressor genes PTEN and RB2.

Refernces:

  1. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nature reviews. Genetics. 2004;5(7):522-31. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15211354.
  2. Wienholds E, Plasterk RH. MicroRNA function in animal development. FEBS letters. 2005;579(26):5911-22. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16111679.
  3. Michael MZ, O’Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific microR-NAs in colorectal neoplasia. Molecular Cancer Research. 2003;1(12):882–891.

Genes: VHL (Von Hippel-Lindau)

  • loss of function is associated with the Von Hippel-Lindau disease
  • Product of the VHL gene is the protein pVHL.
  • Function of pVHL is ubiquitylation of HIF-1α, which is one of two subunits of the HIF-1(hypoxia-inducible factor-1) transcription factor.
  • Polyubiquitylation of HIF-1α leads to its degradation and therefore prevents the formation of a functional HIF-1.
  • A functional HIF-1 transcription factor causes expression of genes such as VEGF, PDGF, TGF-α, which are growth promoting genes in the context of establishing new blood vessels.
  • Normally, pVHL constitutively prevents HIF-1 to become active, but if the cell experiences hypoxia, pVHL fails to bind to the HIF-1α subunit and therefore an adequate stimulation of angiogenesis and erythropoiesis will establish an sufficient oxygen supply for the cells suffering hypoxia.
  • If pVHL cannot form, since VHL was deactivatied by (e.g.) mutation, HIF-1α is constitutively active, which leads to an inappropriate high level of these growth factors, which will favor tumor growth.

Genes: APC (Adenomatous polyposis coli)

Wnt signaling in the small intestine
Wnt signaling in the small intestine
  • Loss of function can contribute to the development of Familial adenomatous polyposis (FAP)
  • Together with GSK-3β and axin Apc builds the β-catenin destruction complex, which playes a key role in the wnt-signalling pathway.
  • In colonic crypts, Apc negatively controls the levels of β-catenin, which governs the proliveration of the enterocytes in the crypt:
    • Cells are produced at the bottom of the crypt and migrate upward to the villus.
    • β-catenin levels decrease coninously from the lower crypt to the upper villus. This is because the migrating cells increase the expression of APC which, in the absence of Wnts, is able to break down β-catenin.
    • The absence of β-catenin drives cells to differentiation.
    • If APC is mutated and therefore not able to build the β-catenin destruction complex, β-catenin levels stay high and prevent cells from differentiation.
    • Continuous proliferation and the formation of polyps is the consequence.

Tumor Suppressor Genes

  • Tumor Suppressor Genes are the counterpart to oncogenes.
  • Inactivation of Tumor Suppressor Genes favours tumor growth.
  • Cancer phenotype is recessive.
    • However, If induced by viruses, cancer phenotype is dominant.
  • Tumor Suppressor Genes can explain the phenomenon of familial cancer syndroms.
  • Tumor Suppressor Genes may be inactivated by LOH (loss of heterozygosity / allelic deletion).
  • LOH occurs at a far higher frequency that mutational alterations. That is why the loss of a TSG is more frequent than somatic mutations (10^6 for one allele, 10^12 for both at the same time).
  • Tumor Suppressor Genes may be inactivated by disabling the promoter.
  • In order to “find” Tumor Suppressor Genes / “find” genes that are not there in the phenotype of interest (cancer), genetic markers that are close to the gene of interest can be used:
    • Restriction fragment length polymorphism: Depends first on SNPs that affect one allele but not the other and second on restriction enzymes that cleave this site.
    • PCR: Also depends on SNPs that affect one allele but not the other: However, in this case there are primers used that are specific to sequences that are polymorphic.
  • Tumor Suppressor Genes are either gatekeepers (direct cells through growth, division and death) or caretakers (maintenance of the genome, genomic stability).
  • Examples for Tumor Suppressor Genes:
    • RB: retinobastoma, osteosarcoma; transcriptional repession, control of E2Fs
    • APC: familial adenomatous polyposis coli; β-catenin degradation ( see also)
    • p53:
    • VHL: Von Hippel-Lindau disease; ubiquitin ligase
    • TGFBR2: TGF-β receptor
    • p16: familial melanoma; CDK inhibitor
    • p14: p53 stabilizer
    • NF1: neurofibromatosis type 1; Ras-GAP
    • NF2: neurofibroma-position syndrome; cytoskeleton-membrane linkage
    • PTEN: breast and gastrointestinal carcinomas; PIP3 phosphatase