Specificity of microRNA target selection in translational repression

MicroRNAs (miRNAs) are a class of noncoding RNAs found in organisms as evolutionarily distant as plants and mammals, yet most of the mRNAs they regulate are unknown. Here we show that the ability of an miRNA to translationally repress a target mRNA is largely dictated by the free energy of binding of the first eight nucleotides in the 5' region of the miRNA. However, G:U wobble base-pairing in this region interferes with activity beyond that predicted on the basis of thermodynamic stability. Furthermore, an mRNA can be simultaneously repressed by more than one miRNA species. The level of repression achieved is dependent on both the amount of mRNA and the amount of available miRNA complexes. Thus, predicted miRNA:mRNA interactions must be viewed in the context of other potential interactions and cellular conditions.     

Identification of coagulation gene 3′UTR variants that are potentially regulated by microRNAs

MicroRNAs have been recognized as critical regulators of gene expression and might affect the risk of venous thrombosis. We aimed to identify 3′ untranslated region (UTR) variants in coagulation genes that influence coagulation factor levels and venous thrombosis risk. The 3′UTR of coagulation genes were sequenced in subjects with extremely high or low plasma levels of these factors in two case‐control studies. In total, 28 variants were identified. Five single nucleotide polymorphisms (SNPs) were predominantly present in one extreme level group (F2 rs1799963, F8 rs1050705 and F11 rs4253429, rs4253430 and rs1062547). Additional to F2 rs1799963, F8 rs1050705 (in men) and F11 rs4253430 were associated with an increased risk of venous thrombosis albeit confidence intervals were wide. The three F11 SNPs were in high linkage disequilibrium with functional variants rs2289252 and rs2036914. Rs1062547 and rs4253430 were associated with a significant increase of plasma FXI activity in heterozygotes and homozygotes in wild‐type controls. In silico prediction revealed that these SNPs might disturb the binding sites of miR‐544 and miR‐513a‐3p. Only miR‐544 provoked a significant decrease of the luciferase activity that was not observed with a rs4253430 mutated vector. In conclusion, these results reinforce that microRNAs are candidates to play a role in haemostasis and complex disorders, such as thrombosis.     

Hidden Genetic Variation in LCA9‐Associated Congenital Blindness Explained by 5′UTR Mutations and Copy‐Number Variations of NMNAT1

Leber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.     

HLA-G haplotype structure shows good conservation between different populations and good correlation with high,normal and low soluble HLA-G expression

HLA-G molecule has considerable impact in various clinical fields, therefore many studies attempted to predict its expression based on HLA-G genotype. These studies have focused on polymorphisms in either the coding region or in one of the two untranslated regions (UTR) of the gene. The aim of our study was to determine if HLA-G haplotype defined based on SNPs 5′ and 3′UTR could be used to predict soluble HLA-G expression in unstimulated individuals. Our findings showed that HLA-G haplotype structure was well conserved between distant populations and that the defined haplotypes were correlated with high, normal and low HLA-G soluble secretors. In conclusion, we showed that this genotyping strategy based on the use of a few selected SNPs rather than isolated SNP analysis allows reliable HLA-G expression in all populations. This strategy could be useful in a number of clinical settings, e.g., predicting graft compatibility immunogenetic laboratories.     

Specific primer amplification of the VP1 region directed by 5′ UTR sequence analysis: Enterovirus testing and identification in clinical samples from hand-foot-and-mouth disease patients

Many genotypes of the enterovirus (EV) pathogens can cause clinical hand-foot-and-mouth disease (HFMD). Therefore, rapid identification and monitoring of HFMD pathogens can be difficult, especially from the original clinical specimens. In this study, both universal pan-enterovirus and EV71/CA16 VP1-specific primer sets were designed and used to examine clinical specimens from HFMD patients. Based on the initial sequence analysis of the 5′-untanslated region (5′-UTR) and VP1 amplification products, additional primers for the VP1 region were redesigned for further genotyping of the remaining small portion non-EV71/non-CA16 specimens. With a known panel, it was possible to identify 15 out of 16 members using 5′-UTR sequence typing and VP1 typing, suggesting good detectability and genotyping of this method. One strain that was not typed by 5′-UTR was shown to be a recombinant virus. When this method was applied to examine clinical specimens from 44 suspected HFMD patients, 41 were detected as EV positive. In only one case, the VP1 sequence could not be identified. Four types of EVs, including CA16 (26/41, 63.4%), EV71-C4 (6/41, 14.6%), CA6 (5/41, 12.2%) and CA10 (3/41, 7.3%), were detected. In conclusion, 5′ UTR amplification sequencing and subsequent VP1 specific primer amplification ensures a high detection rate and good genotyping accuracy in the examination of clinical samples. This detection strategy can be used for routine evaluation and monitoring of HFMD to follow local trends of EV infection.     

Identification of two novel type 1 peroxisomal targeting signals in Arabidopsis thaliana

Peroxisomes lack their own genetic material and must therefore import proteins encoded by genes in the nucleus. Amino acids within these proteins serve as targeting signals: they direct the delivery of the proteins to the organelle. The majority of soluble proteins destined for the peroxisomal matrix utilize a type 1 peroxisomal targeting signal (PTS1): a C-terminal tripeptide that follows the pattern small/basic/hydrophobic. We have discovered two new C-terminal tripeptides that target proteins to peroxisomes in Arabidopsis thaliana. The tripeptides PSL and KRR do not fit the major PTS1 consensus but cause green fluorescent protein to accumulate in peroxisomes of stably transformed Arabidopsis. We have identified forty-one proteins in the Arabidopsis genome that also bear these tripeptides at their C-termini and may therefore be peroxisomal.