Constant Splice-Isoform Ratios in Human Lymphoblastoid Cells Support the Concept of a Splico-Stat

Abstract

Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. . DNA-encoded genetic information is kept separately in the chromosomes of a eukaryotic nucleus and is decoded via RNA intermediates, which are processed and transmitted to their destinations, for example, to the sites of cytoplasmic protein synthesis. Genes in pieces (composed of exons spaced apart by introns) additionally depend on a splice apparatus (spliceosome) that uses splice signals in a primary transcript to recognize exon-intron boundaries (splice sites) and to accurately cut out introns and join exons. Often, splicing generates different mature transcripts from the same gene, a process called alternative splicing (AS). This is achieved by alternative usage of splice sites in precursor RNA transcripts. In this way, the complexity of transcriptomes and proteomes is increased in eukaryotic organisms. Obviously, AS events need control to ensure formation of proper splice forms and ratios.Sequence motifs matching the splice-site consensus are very common in primary transcripts, but only a minute fraction of them are used by the spliceosome. This specificity is achieved by further sequence and structural information within the premature transcript recognized by different proteins. A complex network of highly combinatorial molecular interactions ensures the tissue-, developmental-, and elicitor-specific formation of spliced transcripts and is regulated at multiple points (Hertel 2008;Smith et al. 2008).Millions of short, single-pass cDNA sequence reads have accumulated in databases as expressed sequence tags supplemented from next-generation transcriptome sequencing data, both of which indicate that AS affects almost any multi-exon gene of any mammalian genome (Mortazavi et al. 2008;Wang et al. 2008).Howev...

Alternative 5’ Untranslated Regions Are Involved in Expression Regulation of Human Heme Oxygenase-1

“…Sequence features allocated by MIRb elements can act as upstream transcriptional promoters and often provide functional splice sites [25,26]. We have previously identified extra evidence for MIRb exonization in the human genome with the functional characterization of a novel NOD2 splice-isoform [22]. …”

“…Detection and quantification of minigene derived transcripts was done as previously described [22]. Briefly, PCR amplification was carried out under standardized conditions using Bioline BioMix white (Bioline), one plasmid specific 5’-6-carboxyfluorescein (FAM)-labeled primer (5’-FAM-CTCCGGACTCTAGAGGATC-3’) and one HMOX1 specific primer (5’-ACCTGGCCCTTCTGAAAGTT-3’) according to manufacturer’s instructions.…”

It's a bit over, is that ok? The subtle surplus from tandem alternative splicing

“…The stability of TASS isoform ratios may even suggest a model in which the balance of alternative splice site selection converges to a "defined" set point, a concept termed splico-stat. 11,23 This model does particularly support the finding of highly reproducible isoform ratios in inter-individual comparisons, with an underlying variation of the genetic background which could be expected to cause heterogenity in transregulatory effects. For example, the 5 0 TASS ratio of the Wilms' tumor suppressor gene (WT1) and the 4-isoform pattern of the gene G subunit a S (GNAS; cassette exon plus 3 0 TASS) is surprisingly stable in a large set of different individual mouse and human samples.…”

“…For example, the 5 0 TASS ratio of the Wilms' tumor suppressor gene (WT1) and the 4-isoform pattern of the gene G subunit a S (GNAS; cassette exon plus 3 0 TASS) is surprisingly stable in a large set of different individual mouse and human samples. 23 This is even more surprising given that gene expression, which is supposed to be under tight regulatory control, showed variation over several orders of magnitude between the samples.…”

“…11,23 One example illustrates that TASS may play a role in a homeostatic regulatory network and, related to the stability of the TASS isoform ratio, also has an impact on a clinical phenotype. Usage of 5 0 TASS in Wilm's tumor gene (WT1) results in the insertion/deletion of 9 nucleotides, corresponding to 3 amino acids (KTS).…”