Search Results (5)

The αs2-casein is a phosphoprotein secreted in the milk of most mammals, and it is the most hydrophilic of all caseins. Contrary to genes found in ruminants, in donkeys two different encoding genes for donkey αs2-casein (CSN1S2 I and CSN1S2 II) have been identified. However, unlike in ruminants, the variability at these loci has not been characterized in detail in donkeys until now. In this study, we analyze the transcript profile of the donkey CSN1S2 I and CSN1S2 II genes, and we identify and describe the variability of these loci in the Ragusana and Amiatina breeds reared in Italy. The analysis of the CSN1S2 I Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) products and subsequent sequencing showed, in addition to correctly spliced mRNA, seven other minor mRNAs resulting from differential splicing events involving, in various combinations, entire exons (4, 5, 6, and 11), parts of exons (5′ or 3′ end of exon 17), or the recognition of intronic sequences as an exon (exon 12′). Similarly, the transcription analysis of the CSN1S2 II gene revealed a remarkable variability in splicing events, mainly concerning the alternative insertion of an extra exon 7 (named 7′); the first 33 bp of exon 13; or the alternative skipping of exons 9, 10, 11, 12, and 15, and their combinations. At the mRNA level for CSN1S2 I, seven SNPs were observed, five of which led to amino acid changes: p.T73>A, p.I109>V, p.I130>V, p.I146>T, and p.D217>Y. Similarly, nine SNPs were observed at the CSN1S2 II locus, seven of which are non-synonymous: p.L63>F, p.H70>Q, p.D90>N, p.129A>T, p.H131>Y, p.E144>G, and p.F157>S. In addition, the DNA sequencing of exon 17 and flanking introns of the CSN1S2 I gene revealed a G>A transition at the splice acceptor site of CSN1S2 I exon 17 (FM946022.1:c.375-1G>A), resulting in an allele-specific skipping of the first 15 nucleotides of this exon, which encode the peptide 176NKINQ180, and the recognition of an in-frame cryptic splicing acceptor site: arAACAAAATCAACCAG. A genotyping method based on restriction fragment length polymorphism (XbaI PCR-RFLP) was set up for this SNP. In the total population studied (105 Ragusana and 14 Amiatina donkeys), the A allele had a frequency of 0.2437 with no evidence of deviation from the Hardy–Weinberg equilibrium. This study adds new knowledge regarding the genetic variability of αs2-caseins in donkeys and may contribute significantly to the genetic improvement of milk production for this species. Full article

Alternative splicing (AS) is a major means of post-transcriptional control of gene expression, and provides a dynamic versatility of protein isoforms. Cancer-related AS disorders have diagnostic, prognostic and therapeutic values. Changes in the expression and AS of human stearoyl-CoA desaturase-5 (SCD5) are promising specific tumor markers, although the transcript variants (TVs) of the gene have not yet been confirmed. Our in silico, in vitro and in vivo study focuses on the distribution of SCD5 TVs (A and B) in human tissues, the functionality of the relevant splice sites, and their modulation by certain single-nucleotide variations (SNVs). An order of magnitude higher SCD5A expression was found compared with SCD5B. This unequal splicing is attributed to a weaker recognition of the SCD5B-specific splicing acceptor site, based on predictions confirmed by an optimized minigene assay. The pronounced dominance of SCD5A was largely modified (rs1430176385_A, rs1011850309_A) or even inverted (rs1011850309_C) by natural SNVs at the TV-specific splice sites. Our results provide long missing data on the proportion of SCD5 TVs in human tissues and reveal mutation-driven changes in SCD5 AS, potentially affecting tumor-associated reprogramming of lipid metabolism, thus having prognostic significance, which may be utilized for novel and personalized therapeutic approaches. Full article

X-linked myotubular myopathy (XLMTM) is a severe form of centronuclear myopathy, characterized by generalized weakness and respiratory insufficiency, associated with pathogenic variants in the MTM1 gene. NGS targeted sequencing on the DNA of a three-month-old child affected by XLMTM identified the novel hemizygous MTM1 c.1261-5T>G intronic variant, which interferes with the normal splicing process, generating two different abnormal transcripts simultaneously expressed in the patient’s muscular cells. The first aberrant transcript, induced by the activation of a cryptic splice site in intron 11, includes four intronic nucleotides upstream of exon 12, resulting in a shift in the transcript reading frame and introducing a new premature stop codon in the catalytic domain of the protein (p.Arg421SerfsTer7). The second aberrant MTM1 transcript, due to the lack of recognition of the 3′ acceptor splice site of intron 11 from the spliceosome complex, leads to the complete skipping of exon 12. We expanded the genotypic spectrum of XLMTM underlying the importance of intron–exons boundaries sequencing in male patients affected by XLMTM. Full article

Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing. Full article

One fundamental problem of bioinformatics is the computational recognition of DNA and RNA binding sites. Given a set of short DNA or RNA sequences of equal length such as transcription factor binding sites or RNA splice sites, the task is to learn a pattern from this set that allows the recognition of similar sites in another set of DNA or RNA sequences. Permuted Markov (PM) models and permuted variable length Markov (PVLM) models are two powerful models for this task, but the problem of finding an optimal PM model or PVLM model is NP-hard. While the problem of finding an optimal PM model or PVLM model of order one is equivalent to the traveling salesman problem (TSP), the problem of finding an optimal PM model or PVLM model of order two is equivalent to the quadratic TSP (QTSP). Several exact algorithms exist for solving the QTSP, but it is unclear if these algorithms are capable of solving QTSP instances resulting from RNA splice sites of at least 150 base pairs in a reasonable time frame. Here, we investigate the performance of three exact algorithms for solving the QTSP for ten datasets of splice acceptor sites and splice donor sites of five different species and find that one of these algorithms is capable of solving QTSP instances of up to 200 base pairs with a running time of less than two days. Full article