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RNA interference mechanism and applications

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... 'The RNA World' was coined by Walter Gilbert in 1986 in a commentary on the then ... Gilbert pointed out that neither DNA nor protein were required in such a ... – PowerPoint PPT presentation

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Title: RNA interference mechanism and applications


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RNA interference mechanism and applications Micro
RNA, RNAi, and SiRNA
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Cell. 1993 Dec 375(5)843-54 The C. elegans
heterochronic gene lin-4 encodes small RNAs with
antisense complementarity to lin-14.Lee RC,
Feinbaum RL, Ambros V.Harvard University,
Department of Cellular and Developmental Biology,
Cambridge, Massachusetts 02138. lin-4 is
essential for the normal temporal control of
diverse postembryonic developmental events in C.
elegans. lin-4 acts by negatively regulating the
level of LIN-14 protein, creating a temporal
decrease in LIN-14 protein starting in the first
larval stage (L1). We have cloned the C. elegans
lin-4 locus by chromosomal walking and
transformation rescue. We used the C. elegans
clone to isolate the gene from three other
Caenorhabditis species all four Caenorhabditis
clones functionally rescue the lin-4 null allele
of C. elegans. Comparison of the lin-4 genomic
sequence from these four species and
site-directed mutagenesis of potential open
reading frames indicated that lin-4 does not
encode a protein. Two small lin-4 transcripts of
approximately 22 and 61 nt were identified in C.
elegans and found to contain sequences
complementary to a repeated sequence element in
the 3' untranslated region (UTR) of lin-14 mRNA,
suggesting that lin-4 regulates lin-14
translation via an antisense RNA-RNA interaction.
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miRNA genes
  • Most miRNA genes come from regions of the genome
    quite distant from previously annotated genes,
    implying they derive from different transcription
    units (TUs)
  • The miRNAs within a genomic cluster are often
    related to each other (but not always)
  • Not all of the cloned miRNAs are conserved even
    in very closely related animals, such as
    human/mouse, C. elegans/C. briggsae

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The actions of small silencing RNA
A, mRNA cleavage specified by a miRNA/siRNA B,
translational repression specified by
miRNAs/siRNAs C, transcriptional silencing,
thought to be specified by heterochromatic siRNAs
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Computational program to identify miRNA genes
  • Significant progress has been made in miRNA
    research since the report of the lin-4 RNA(1993).
    About 300 miRNAs have been identified in
    different organisms to date.
  • However, experimental identification miRNAs is
    still slow since some miRNAs are difficult to
    isolate by cloning due to low abundance
    /stability/ expression pattern/cloning procedure.
    Thus, computational identification of miRNAs from
    genomic sequences provide a valuable complement
    to cloning.

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MiRNA today
Number of miRNA found Computational approach
experimentally validated Human200-255
175 Nematods103-120
100 Flies96-124
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miRNA functions Fliescontrol of cell
proliferation, cell death and fat
metabolism Nematods neuronal patterning and
embryonic development. Mammals hematopoietic
lineage differentiation. Plants flower
development, leaf development and axial meristem
initiation
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