Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons

Abstract

ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3′ mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.

Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene

“…Moreover, being the good basis to generate isogenic differentiation lineages, they provide an excellent in vitro platform to study the phenotype of DGCR8 deficiency in several human somatic cell types. Since one consequence of lower DGCR8 expression is the suppressed maturation of canonical miRNAs, the use of our special hESC line can also enhance the studies of mirtrons, a special class of Microprocessor-independent but splicing-dependent miRNAs [ 54 , 55 , 56 , 57 , 58 , 59 ]. Taken together, this DGCR8 +/− cell line can well-contribute to the study of the dominant human RNA interference pathway, as well as to the better understanding of the “noncanonical” functions of the DGCR8 protein.…”