Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products

Abstract

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

Inhibition of Rho Kinases Enhances the Degradation of Mutant Huntingtin

“…The N terminus fragment of AR99Q, was amplified by PCR using primers BglII-AR-Fw and SalI-AR-396Rv (AAAAAAGT-CGACGACGCAACCTCTCTCGGGGT), cleaved by BglII and SalI, and subcloned into the BglII-SalI sites of the pEGFP-C1. EGFP-DRPLA construct encoding atrophin-1 with Gln-71 was described previously (39) and was kindly provided by Dr. Masao Yamada. To prepare pcDNA3.1-tNhtt-60Q-EGFP for transient transfection, tNhtt-polyQ-EGFP fragment was cut from pINDtNhtt-polyQ-EGFP (40) with HindIII-XbaI digestion, and the resulting fragment was inserted into pcDNA3.1-v5/His plasmid.…”

Assessing the fraction of short-distance tandem splice sites under purifying selection

“…Apart from experimental investigations (Condorelli et al 1994;Vogan et al 1996; Burgar et al 2002;Tadokoro et al 2005), another approach to address this question is to estimate the fraction of tandem sites under purifying selection. Here, we show that the sequence conservation level differs between tandem motifs due to constraints on the splice site consensus and possibly on splicing enhancer motifs as well as on the coding sequence.…”

“…Although tissue-or cell-line-specific splicing has been observed at tandem acceptors (Koenig Merediz et al 2000;Hiller et al 2004;Xu et al 2004;Tadokoro et al 2005) and tandem donors (Hu et al 1999;Yan et al 2000), stochastic selection of either of the two splice sites likely explains alternative splicing at most tandems (Chern et al 2006). Stochastic splice events are expected to yield similar splice variant ratios in different tissues, and this was observed in many cases (Vogan et al 1996;Hammes et al 2001;Burgar et al 2002;Tadokoro et al 2005;Hiller et al 2006b).…”

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

“…Many of these sites are 3 nucleotides apart and result in inclusion or exclusion of a single codon. There is debate about the nature of splicing regulation at these sites 5,6 with some arguing that the splicing motif differences are so subtle that selection is stochastic 7 , while others infer regulation based on tissue specificity 8 .…”

“…Tandem splice site selection has been analyzed semi-quantitatively using modified capillary electrophoresis 7 , and high-resolution gel electrophoresis 8 . RNA-Seq (RNA sequencing) reads can be used to quantify the splicing ratios at each splice site.…”