Bajracharya J et al. International Journal of Medicine and Biomedical Sciences.

2016; 1(4):17-21

ORIGINAL ARTICLE ISSN: 2467-9151 OPEN

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In silico Validation of Expressed Sequence Tags for Alternate Splice Variants of

Human myo-inositol Oxygenase Gene
Jayendra Bajracharya1*, Manoj Pradhan2, Anju Bajracharya3
1
Department of Biochemistry, Nepalese Army Institute of Health Sciences
2
Department of Microbiology, Nepalese Army Institute of Health Sciences
3
a
Department of Pharmacy, Little Angels College of Higher Studies, JF Institute of Health Sciences

ABSTRACT
myo-Inositol oxygenase (MIOX) is an enzyme that catabolizes myo-inositol in humans. There is increased MIOX
expression and increased MIOX activity in renal tubular cells in diabetic nephropathy. One potential mechanism of
increased expression and activity of MIOX is the production of one or more highly stable alternative splice isoform of
MIOX transcript. Based on the evidence gathered from the expressed sequence tags (ESTs) associated with MIOX
protein sequence, we report that retention of intron 9 in MIOX transcript is a valid alternative splice mechanism. This
alternative splice isoform is predicted to have higher stability than the canonical MIOX transcript. Higher stability of the
alternative splice variant leads to increased production of MIOX protein isoform with retained structural and potentially,
functional features of canonical MIOX protein. Production of this MIOX transcript isoform could be the mechanism of
increased expression and activity of MIOX in renal tissues affected by diabetes mellitus. Alteration of the splice
mechanism could be a new therapeutic target in prevention and treatment of complications of diabetes mellitus.
Keywords: Alternate splice variants, myo-inositol oxygenase, MIOX, expressed sequence tag, In silico validation

INTRODUCTION
myo-Inositol oxygenase (MIOX) is an enzyme involved in
Alternative splicing of pre-mRNA involves a complex
the myo-inositol catabolic pathway in humans [1]. The
machine called splicesome, which recognizes the donor
human MIOX gene is composed of 3538 nucleotides with
(GU) and acceptor (AG) splice site signals at 5’ and 3’ends
ten exons and nine introns. The MIOX gene is transcribed
of an intron respectively. Once the signals are read,
to an 855-nucleotide mature transcript. MIOX is expressed
excision of the pre-mRNA occurs at these sites. The
mainly in the cells of the proximal convoluted tubule [2].
excised segment (intron) forms a lariat while the exons are
There is growing evidence that implicate the roles of
glued together to form a mature mRNA [4]. Alternative
increased MIOX expression and consequently, its activity
splicing can involve exon skipping, complete or partial
in renal tubular cells in pathogenesis of diabetic
intron retention, and alternate splice site acceptor or donor
nephropathy [3]. A variety of factors modulate the
at the 5' or the 3' ends within any of the exons or introns.
expression of a gene. Alternative splicing, however, can
Identification of the alternative splice variants of
modulate the stability of the transcript and ultimately,
transcripts require examination of database of expressed
affect the activity of the protein product [4]. Increase in
sequence tags (ESTs), which are short cDNA sequences
MIOX expression and activity in renal tubular cells in
that represent the expression of a gene in a specific tissue.
diabetes may well be related with formation of highly
Detailed analysis of the putative protein products of the
stable isoforms of MIOX transcripts by alternative
alternative splice variants of MIOX transcript is required
splicing.
to validate the ESTs.
*Correspondence: Jayendra Bajracharya
Department of Biochemistry, Nepalese Army Institute of
Health Sciences
E-mail: [email protected]

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METHODOLOGY sources, and percent identities of the ESTs with intron 9 of

MIOX gene are shown in Table 2. Based on the higher
The MIOX protein was used as a query sequence for percent identity with intron 9 of MIOX gene, it can be
TBLASTN [5] search of ESTs in the database of expressed concluded that retention of intron 9 is a highly likely
sequence tag [6] (dbEST) archived in EST divisions of alternative splice event for MIOX gene.
GenBank - the genetic sequence database of National
Institute of Health (NIH), European Molecular Biology Table 1. Alternative splice events observed in ESTs of
Laboratory (EMBL), and DNA Data Bank of Japan MIOX
(DDBJ). The search was carried out on July 16, 2016 at
12:11 AM NST.
Accession number
Once the ESTs were retrieved, local alignment tools such
as CLUSTAL W [7] and Spidey [8] were used to align Alternative splice events of
each of the EST with MIOX gene and its mature transcript ESTs
for locating the exons, introns, untranslated regions and AI493089
splice site signals on the ESTs. AI632704
Near complete retention of
AI650911
Once the alternative splicing events were identified, virtual intron 9
AI820659
mRNAs were constructed in silico by incorporating the
alternate splice event to the MIOX canonical transcript. AW188505
The stability of the major alternate splice variant was Near complete retentions of
examined by a program called RNAfold [9]. The intron AI792051
secondary structures and the free energies of these variants 7 and intron 8
were also compared to that of MIOX canonical transcript. Near complete retention of
AW243258
intron 8
These alternate splice variants were translated by using
various translation programs such as Transeq and Partial retention of intron 1 at
CB956269
Translator. The predicted protein products were then 5' end
aligned with the canonical MIOX protein using Partial retention of intron 5
CLUSTALW. PredictProtein [10] and Swiss Model [11] DA621131
at 3' end
were used to examine the structure of the predicted protein Partial retention of intron 9 at AW300006
products.
3' end BE463728
BG431276
Skipping of exon 3
BI850744
RESULT Alternate splice acceptor at BI517843
3' end of exon 6 BI518138
The TBLASTN search carried out using MIOX protein Alternate splice acceptor at AI434607
sequence as a query against dbEST returned 96 hits. Upon 3' end of exon 9 BF590438
careful examination of the 94 hits, there were 75
Alternate splice donor at AI580367
independent ESTs. Some ESTs were probably repeated in
the hits due to alignment of those ESTs with multiple parts 5' end of exon 9 BM924395
of the MIOX protein. Out of the 75 independent ESTs, 53 Alternated splice donor at
BG165729
ESTs were found to have either normal splicing or 3' end of intron 4
truncated parts of the mature canonical transcript of Multiple alternate splice events AW194937
MIOX. Twenty-two ESTs showed some form of
alternative splice events, which is summarized in Table 1.

Of the different alternative splice events, near complete

retention of intron 9 was evident in five independent ESTs. It is evident that the ESTs did not perfectly align with
Other splice events were observed in fewer ESTs. intron 9 sequence throughout its length as shown in Figure
1. This is much more likely due to sequencing error of the
Intron 9 is 103 nucleotides in length. As there are five transcripts. The presence of very few alternative splice site
independent ESTs, near complete or possibly, complete signals within intron 9 possibly means that the alternative
retention of intron 9 is more likely to be a plausible splice variant of MIOX gene most likely would contain the
alternative splice event for MIOX gene. The lengths, complete sequence intron 9.

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Figure 1. Multiple sequence alignment of the ESTs with near Figure 2. Secondary structures of canonical transcript of
complete retention of intron 9 of MIOX gene as aligned with MIOX (top) and the putative transcript of MIOX with intron
the intron 9 sequence 9 retention (bottom).

Moreover, the secondary structure of in silico generated Free energies of the mRNA secondary structures determine
virtual mRNA constructed by incorporating the complete their stabilities. Free-energy calculations of the ESTs and
sequence of intron 9 was found to be similar to the native transcript show that native mRNA sequences have
secondary structure of MIOX canonical transcript as more negative free energy than randomized mRNA
shown in Figure 2. sequences with similar nucleotide compositions (Seffens
and Digby, 1992). As predicted by the RNAfold program,
the intron 9 complete retention splice variant mRNA has
the most negative free energy (-725.15 kcal/mol) while the
MIOX canonical transcript has a negative free energy (-
655.90 kcal/mol). Thermodynamically, low free energy
means higher stability. So, the alternative splice variant
with complete incorporation of intron 9 in MIOX mRNA
is highly likely to be stable, and thus, may be transcribed
to a functional protein.

In silico translation of the alternative splice variant with

complete incorporation of intron 9 in MIOX mRNA
produced a protein product of 304 amino acids. As the
numbers of nucleotides added to make these splice variants
is 103, which is not a multiple of three, frame-shift is
predicted to occur downstream of this sequence. The
translated product retains high identity to the canonical
MIOX protein and also retains all the secondary structures
of canonical MIOX protein. Modeling of the active site of
the translated product shows the retention of the MIOX
active site in this protein as shown in Figure 3.

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The molecular alterations and mechanisms that lead to

retention of intron 9 in MIOX transcript is an area that
needs to be explored. Isolation of the protein product of the
splice variant is needed to confirm the in vivo existence of
the splice variant. Assays for enzyme activity will also be
needed to determine the actual enzymatic activity of the
protein product. If in vitro and in vivo studies prove that
the retention of intron 9 in MIOX transcript leads to
increased MIOX activity in tissues affected by diabetes
mellitus, this could direct the current research to
development of a new therapeutic strategy on preventing
and/or treating the complications of diabetes mellitus.
Figure 3. Homology modeling of MIOX protein isoform
produced from retention of intron 9 is shown on the right.
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