1. In vitroIn vitro transcription:translation of alternatively spliced calpastatintranscription:translation of alternatively spliced calpastatin cDNAscDNAs
KirstyKirsty K Jewell, ChristopherK Jewell, Christopher PrevattPrevatt, Tim Parr, Paul L, Tim Parr, Paul L SenskySensky, Ronald G Bardsley and Peter J Buttery, Ronald G Bardsley and Peter J Buttery
Division of Nutritional Biochemistry, School of Biosciences, UniDivision of Nutritional Biochemistry, School of Biosciences, University of Nottingham, Suttonversity of Nottingham, Sutton BoningtonBonington Campus, Loughborough, LE12 5RD, UKCampus, Loughborough, LE12 5RD, UK
IntroductionIntroduction
Calpastatin is an endogenous inhibitor of the intracellular calpain proteinases that are active
during cytoskeletal remodelling. Calpastatin mRNA transcripts (Types I-III) are produced by
alternative promoter usage and alternative splicing from a single gene (Figure 1). Based on
mRNA sequences, Type I-III proteins would have variable XL and L N-terminal leader
sequences, which have no inhibitory function but may be involved in intracellular targetting.
However, it has not so far been demonstrated that all mRNA variants are actually translatable.
Although all mRNA transcripts are present both in heart and skeletal muscle, in previous work
we have found that SDS PAGE indicates protein bands of approximately 130-145kDa (heart) or
130kDa only (muscle). These Mr are difficult to interpret because calpastatin peptides travel
anomalously on SDS PAGE. Furthermore the Type I and Type II transcripts each have two
potential in-frame translation start sites in exons 1xa/exon 2 and in exons 1xb/exon 2
respectively, whereas Type III has only one site in exon 2 (Figure 2). It is not clear whether
the upstream or downstream AUG or both are used during translation of Types I and II mRNA.
Figure 1. Calpastatin protein domains, gene structure and splice patterns
MethodsMethods
• A series of full length calpastatin cDNAs (Figure 2) was generated by RT PCR from heart and
skeletal muscle RNA
• Constructs were cloned into the pTNT vector (Promega) for in vitro transcription/translation
in the presence of 35S-Met. 5’UTR are not included in these constructs.
• A parallel series of constructs was produced by site-directed mutagenesis via the QuikChange
procedure (Stratagene) to replace the downstream ATG in exon 2 (M→K, confirmed by DNA
sequencing)
• In vitro transcription:translation products were analysed by 8% SDS PAGE and
autoradiography (Figure 3) Results and ConclusionResults and Conclusion
Types I and II generated a range of bands between ~145 and ~120 kDa
bands, whereas the Type III construct generated a ~128kDa band only (Figure
2), suggesting that both upstream and downstream ATG could be utilised in
Types I and II. Proteins derived from constructs lacking exon 3 (∆3),
characteristic of skeletal muscle, had slightly increased mobility, but removal of
exon 3 did not account for the 145/120 kDa differential. Removal of the
downstream ATG in Type III prevented its translation, but did not prevent the
formation of 124-132kDa peptides from Types I and II which could therefore
result from proteolytic truncation. Surprisingly, a combined deletion of the
downstream ATG and exon3 completely eliminated translation from Type I,
which should have retained a functional upstream ATG. Since exon 3 is usually
deleted in skeletal muscle, this may be evidence of a tissue-specific
translational control mechanism.
We thank BBSRC for financial support and studentship for CP
Type I
Type II
Type III
Type III ∆3
Type I ∆3
promoter
1u 3 4 30+
XL L II III IVI
protein
gene
Figure 2. Calpastatin cDNA constructs in pTNT vector (Promega)
mutType I
mutType I ∆3
mutType III
mutType III ∆3
mutType II
ATG ATG
Type I Type I
∆∆∆∆3
Mut
Type I
Mut
Type I
∆∆∆∆3
Mut
Type II Type
III
Type III
∆∆∆∆3
Mut
Type III
Mut
Type III
∆∆∆∆3
142
132
130
124
Figure 3. In vitro transcription:translation products from pTNT constructs (Figure 2)
kDa
1y 1z
ATG ATG ATG
Type II
5’UTR
1xa
1xb
2