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Experimental validation of a predicted microRNA within human FVIII gene.

ABSTRACT: Hemophilia A is an X-linked bleeding disorder that occurs due to the deficiency of Factor VIII (FVIII) protein clotting activity. The mutations in the F8 gene, which encodes FVIII coagulating protein have been widely reviewed. However, there is a wide range of criteria that in addition to F8 gene mutations, different molecular mechanisms may be associated with hemophilia A. Various functions of FVIII could be related to the hypothetical small non-coding RNAs, located within the F8 gene sequence. Therefore, miRNAs that can post-transcriptionally regulate gene expression might confer susceptibility to developing hemophilia A. Here, we have selected a bioinformatically predicted hairpin structure sequence in the first intron of the F8 gene that has the potential to produce a real miRNA (named put-miR1). We tried to experimentally detect the predicted miRNA via RT-PCR following its precursor overexpression in HEK 293 cell lines. Despite the accuracy of miRNA prediction, according to the reliable bioinformatics studies, we couldn't confirm the existence of considered mature miRNA in transfected cells. We hope that through changing experimental conditions, designing new primers, or altering cell lines and expression vectors, the exogenous and endogenous expression of the predicted miRNA will be confirmed.

SUBMITTER: Jalali-Qomi S

PROVIDER: S-EPMC8310658 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Experimental validation of a predicted microRNA within human FVIII gene.

Jalali-Qomi Shahabeddin S Motovali-Bashi Majid M Rezaei Halimeh H Khalilian Sheyda S

Molecular biology research communications 20210601 2

Hemophilia A is an X-linked bleeding disorder that occurs due to the deficiency of Factor VIII (FVIII) protein clotting activity. The mutations in the F8 gene, which encodes FVIII coagulating protein have been widely reviewed. However, there is a wide range of criteria that in addition to F8 gene mutations, different molecular mechanisms may be associated with hemophilia A. Various functions of FVIII could be related to the hypothetical small non-coding RNAs, located within the ...[more]

PMID: 34316492

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Project description:Computational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations.We have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines.The experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.

Dataset Information

Experimental validation of a predicted microRNA within human FVIII gene.

Publications

Experimental validation of a predicted microRNA within human <i>FVIII</i> gene.

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