U2hr Mutation Caused By Marie Unna Type Of The Molecular Mechanisms Of Genetic Dilute Hirsutism

Hair growth and shedding occur periodically,characterized by cyclic transformation consisting of phases of rapid growth(anagen),apoptosis-driven regression(catagen),and relative quiescence(telogen).Hair loss and unwanted hair growth(hypertrichosis) emerge owing to disturbance of hair cycle.Marie Unna hereditary hypotrichosis(MUHH) is an autosomal dominant hair loss disorder,with the affected individuals usually born with little or no hair,and coarse twisted hair growth on the scalp in early childhood.In most patients,scalp hair is lost progressively,starting at puberty or beyond.Our previous research and several other studies have mapped the MUHH gene to chromosome 8p21. The mutational screening of the Hairless(HR) gene,which lies in the positioned region and plays a pivotal role in the hair cycling process,however,found no pathogenic change in the coding region.We previously discovered 13 mutations centerlized in the second upstream open reading frame(uORF)(designated as U2HR) region,which is one of the four uORFs(refer to as U1HR,U2HR,U3HR and U4HR,respectively) in the 5'-untranslated region(UTR) of the HR gene.Seven mutations,which consist of mutations in initiation codon or termination codon,as well as one nonsense mutation and four different missense mutations,were chosen for this study to investigate their effects on the downstream HR gene expression regulation.Then we attempted to explore the underlying molecular mechanism regulating the HR gene expression via U2HR.Cis-acting elements in 5'-UTR generally have negative regulation on expression of downstream cistron.Our previous studies have ruled out the potential effects of U3HR and U4HR on the transcriptional and translational level of downstream reporter genes, whereas disruption of initiation codon in U1HR or U2HR significantly reduced(U1HR) or increased(U2HR) the translation efficiency with nearly no effects on the transcriptional level,suggesting that U1HR and U2HR function as stimulatory and inhibitory translational control elements,respectively.Additionally,we discovered dramatic increase(2～4 folds versus wildtype(WT)) of translational level of reporter genes(Luciferase and EGFP) after transfection of the above seven mutant constructs into HeLa cell line.We found no effects on reporter gene transcriptional level when compared with WT.These findings helped us realize that loss-of-function mutations in U2HR could lead to increased main ORF(mORF) translation of the HR gene,and the underlying mechanism causing MUHH is over-expression of the HR gene.Inhibitory uORFs may function in nucleotide sequence dependent or nascent peptide sequence dependent way.We generated three synonymous mutants at the C terminal region of U2HR coding sequcence,and no obvious changes were found in the translational level of Luciferase or EGFP reporter gene.This indicated that the translation regulation of HR gene depends on the amino acids encoded from U2HR coding sequence.We performed site-directed mutagenesis to remove the termination codons of U1HR and U2HR and allow the fusion of EGFP sequence.The observation of efficient translation of the two uORFs in transfected cells,as well as verification of the translation of U2HR using a polyclonal antibody to U2HR,lead us to believe that the functional U1HR and U2HR seem to encode active peptides.In the complementary test,over-expression of U2HR peptide could not recover or strengthen the inhibitory effect of U2HR on the HR gene mORF translation,indicating that translation regulation of the HR gene mORF via U2HR is the consequence of cis-acting effect.To further determine whether each amino acid derived from U2HR coding sequence is necessary to regulate downstream mORF expression,we randomly chosed two codons in the N terminal region of U2HR to generate two missense mutants.In the following reporter gene detection,we found that these two mutants increased the translation level of reporter genes by around 2 folds,indicating that N terminal region of U2HR may play the similar role as C terminal region in downstream mORF translation regulation.Stalling at a codon in a uORF can strongly impact downstream translation.Thus,we used site-directed mutagenesis to lengthen the U2HR from 34 to 49 codons so as to alter the amino acid context around the WT U2HR termination codon.This mutant didn't influence the translation level of the reporter genes,indicating no effects on the scanning ribosomes to move downstream.We then inserted a single nucleotide G into the immediate downstream of U2HR initiation codon,causing frame shift mutation and lengthening the codons up to 49.This mutant thus generally altered the context of amino acids of U2HR.We discovered no obvious effect of such mutant on the transcription activity of reporter gene,whereas up to 2 folds increase of the translation level was detected,consistent with mechanism of nascent peptide sequence dependent translation regulation.If the U2HR peptide is translated in physical condition,it may influence the movement of scanning ribosomes via interactions with a certain element in the translation machinery. eIF2a,eIF2b,eIF4e and ERF1 were selected as candidates to verify their possible respective interaction with U2HR peptide via yeast two-hybrid system and mammalian two-hybrid system,but the negative results indicated no interaction between U2HR peptide and a certain candidate protein occur.In summary,these results suggested that the expression level of HR protein in physical condition might be strictly regulated via U2HR.The loss-of-function mutation of U2HR gave rise to over expression of HR gene,and thus caused MUHH.These findings uncover the regulation mechanism underlying HR gene expression,and provide molecular basis for the pathogenesis of MUHH.