Characterization and knockdown of zebrafish hnRNP A1

Mr Imam Cartealy (2008). Characterization and knockdown of zebrafish hnRNP A1 MPhil Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author Mr Imam Cartealy
Thesis Title Characterization and knockdown of zebrafish hnRNP A1
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2008-04
Thesis type MPhil Thesis
Supervisor Ross Smith
Joe Rothnagel
Total pages 103
Total colour pages 15
Total black and white pages 88
Subjects 270000 Biological Sciences
Formatted abstract Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA binding proteins
that form complexes with RNA polymerase II transcripts. The hnRNPs are among the
most abundant proteins in eukaryotic cell nuclei. A subfamily of the hnRNPs, hnRNPs
A/B, which consists of hnRNPs A1, A2 and A3 is characterized by the presence of
tandem RNA Recognition Motifs (RRMs) at the amino terminus, linked to a C-terminal
Gly-rich domain (GRD).
One of the best characterized hnRNP A/B proteins, hnRNP A1, has been
identified in many different aspects of cell metabolism such as alternative splicing of
mRNA, nucleocytoplasmic transport of mRNA, and maintenance of telomere ends.
Using a bioinformatic approach, we have identified a zebrafish gene with high
nucleotide sequence identity to human hnRNP A1. This cDNA was subsequently
cloned and characterized as zebrafish hnRNP A1. The full-length zebrafish cDNA
clone is 1560 bp in length with an open reading frame of 1212 bp. The 5'- and 3'- UTRs
of zebrafish hnRNP A1 are 50 and 298 bp, respectively. Comparison of corresponding
exons and introns between the human and zebrafish hnRNP A1 genes revealed a similar
overall organization. These are: the first exons of both sequences encode only a few
amino acids; the tandem RRM region is encoded by 4 exons with the conserved
ribonucleoprotein consensus sequence 1 and 2 (RNP CS or RNP) in the first RRM
encoded by two separate exons whereas in the second RRM they are encoded by a
single exon; the GRD is encoded by 6 separate exons; the penultimate exon contains thestop codon and the last exon encodes the 3'UTR.
At the protein level, most features of human hnRNP A1 are conserved in
zebrafish hnRNP A1, especially features that are commonly found in the tandem RRM
domain. For example, RNP1 and RNP2, which are responsible for the alternative
splicing and nucleic acid binding activity of hnRNP A1.
Southern blot experiments were performed in order to identify the copy number
of the hnRNP A1 gene that exists in zebrafish. The result indicated that hnRNP A1 gene
presents as a single copy in the zebrafish genome. Northern blot experiments were also
performed to determine the hnRNP A1 mRNA transcript since it was alternatively
spliced in human to give rise to two isoforms. The experiments showed a single
transcript of hnRNP A1 mRNA.
The expression level of zebrafish hnRNP A1 during the development stages was
determined using semi-quantitative RT PCR. This indicated that the expression level is
approximately constant from embryogenesis up to adult stages. In the embryos, the
hnRNP A1 mRNA was ubiquitously expressed in the animal pole of the embryos during
the first 10 hours post fertilisation (hpf). At a later stage (24 hpf), the expression was
more localized to brain and spinal cord neurons. In the brain, the hnRNP A1 can be
found in the forebrain, midbrain and hindbrain.
In order to knock down the expression hnRNP A1, morpholino oligonucleotides
were used. Two morpholino oligonucleotides, one designed against the 5'UTR and the
other against the translational start site, were used in this experiment. Both of themorpholinos produced similar phenotypes, which confirmed the specificity of the
morpholinos in knocking down the expression of hnRNPA1. However, the two
morpholino oligonucleotides showed different activities with the one against the start
codon having less effect on the embryos, resulting in fewer affected embryos. Three
classes of morphant were observed. Class I was identified by small or undeveloped
embryos. Class II had a kinked body axis or disorganized somites. Class III was
characterized by the disorganization in the brain, especially by the lack of a midbrainhindbrain
boundary.
The expression patterns of hnRNP A1, pax6, hlx, dlx2, pax2.1 and emx were
unaltered by the A1 morpholinos. However, the expression pattern of shh and slit1a
were altered by the A1 morpholinos injection and showed a “kink” pattern of floor
plate. The “kink” pattern shown by slit1a or shh indicates that the changes of the
expression pattern could cause the morphological change. Alternatively, the
morphological changes could affect the cell population in this region, thereby changing
the mRNA levels and distribution.
Additional Notes 45, 49, 51-52, 60, 67, 69-71, 73-75, 77-79 lanscape : 47

 
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Created: Thu, 23 Oct 2008, 13:31:21 EST by Mr Imam Cartealy