MicroRNA-Directed Cleavage of HOXB8 mRNA

1
MicroRNA-Directed Cleavage of HOXB8 mRNA

• Soraya Yekta,I-hung Shih, David P.Bartel
• 23 APRIL 2004 VOL 304 SCIENCE

2
Hox genes in mammals

• The vertebrate homeotic complex comprises four
  distinct Hox gene clusters (Hox A, B, C, D) that
  are organized into thirteen homology
• The chromosomal organization of the genes in each
  Hox cluster reflects its anterior-posterior
  expression in the body plan (spatial
  colinearity).
• In general, members of the same subgroup are
  expressed at the same time and have the same
  anterior boundary of expression

3
The current model for the biogenesis and
post-transcriptional
suppression of microRNAs and siRNAs
4
Recent progress

• In plant, most miRNAs anneal to their targets
  with nearly complete complementarity at a single
  site,leading to mRNAs cleavage and degradation
• The extent of complementarity between the
  siRNA/miRNA and its target can determine the
  mechanism of silencing

5
miR-196 in Vertebrate
6
The sequence and genomic context of the
vertebrate miR-196 family
Bold residues of the mature miRNA Red miRNAs
validated by cloning. Asterisks perfectly
conserved residues
7
Overview of the 5 RACE Procedure
8
RNAse protection assay
9
Expression of miR-196 during mouse embryonic
development.
The Northern blot probes total RNA from 7-day,
11-day, 15-day, and 17-day mouse embryos (lanes
14, respectively) M 33P-labeled RNA size
markers
10
MiR-196directed cleavage of HOXB8 mRNA(1)
black Absolutely conserved nucleotides red
complementary site pink GU wobble
11
MiR-196directed cleavage of HOXB8 mRNA(2)
perfect antisense sequence of miR-196a
miR-196a-as perfect antisense sequence of
miR-1d miR-1d-as miR-1d expressed in skeletal
muscle cells

12
MiR-196directed cleavage of HOXB8 mRNA(3)

13
Additional HOX genes predicted to be
regulatory targets of miR-196(1)
Wide red bars extensive complementarity, narrow
bars seed matches, Asterisks slightly
relaxed seed matches that permit GU wobble
14
Sequence alignment of the miR-196 complementary
site, and a representative complementary seed and
flanking regions in the 3 UTRs of HOXC8 and
HOXD8 genes.
Blackabsolutely conserved nucleotides Red
miR-196 complementary site and the seed PinkGU
wobbles
15
Additional HOX genes predicted to be
regulatory targets of miR-196(2)
16
Additional HOX genes predicted to be
regulatory targets of miR-196(3)
17
Discussion

• Transcriptional repression may also involved
• Genetic model of mRNA cleavage in vivo
• The difference of miRNA and siRNA
• The mechanism of miRNA function

18
Thank you!