Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast

Specificity of RNA binding by the Mmi1 YTH domain. (A) RNA versus DNA recognition. The nucleic acid binding reaction mixtures contained 100 nM 5′ ³²P-labeled 14-mer DSR RNA or 14-mer DSR DNA (shown at bottom) and 125, 250, 500, or 1000 nM Mmi1(319–488). Mmi1-YTH was omitted from the control reactions in lanes indicated by (–). (B) DSR RNA hexanucleotide recognition. The RNA binding reaction mixtures contained 100 nM 5′ ³²P-labeled 14-mer RNA with a consensus DSR hexamer UUAAAC (left panel) or 14-mer RNA with CCGGGU in lieu of the consensus DSR (right panel) and 125, 250, 500, or 1000 nM Mmi1(319–488). (C) Variant DSR recognition. RNA binding reaction mixtures contained 100 nM 5′ ³²P-labeled 14-mer RNA with a consensus DSR hexamer UUAAAC (left panel) or 14-mer RNA with a variant UUAAAU hexamer (right panel) and 125, 250, 500, or 1000 nM Mmi1(319–488). Mmi1(319–488) was omitted from the control reactions in lanes indicated by (–). The free nucleic acids and nucleic acid•protein complexes were resolved by native PAGE and visualized by autoradiography.