Journal: Nucleic Acids Research

Article Title: A specialized TFIIB is required for transcription of transposon-targeting noncoding RNAs

doi: 10.1093/nar/gkaf427

Figure Lengend Snippet: Tetrahymena life cycle and the identification of Emit3. ( A ) Tetrahymena cells contain two distinct types of nuclei: the somatic macronucleus (MAC) and the germline micronucleus (MIC). The MAC has continuous transcriptional activity, while the MIC remains transcriptionally inactive until meiosis begins. TEs (depicted in magenta) are predominantly retained in the MIC but eliminated from the new MAC. During conjugation, when two cells of different mating types pair, TE-targeting ncRNAs are synthesized in the meiotic MIC. Meiotic prophase is categorized based on the extent of nuclear elongation. In stages II–IV, centromeres (Cen.) and telomeres (Tel.) are clustered at opposite poles of the elongating MIC. By stage V, these structures detach from the MIC termini, although telomeres remain clustered. The ncRNA transcription (indicated by dots) is initially concentrated in IES-rich telomeric and pericentromeric regions during stages II and III. In stages IV and V, transcription is restricted to a narrow telomeric region. ( B ) Current knowledge about the MIC ncRNA transcription activation mechanism. See the text for details. ( C ) MIC ncRNA transcription activators and meiosis regulators were grouped into different clusters with respect to the timing of gene expression. Hpm: Hours post mixing. ( D ) Upper panel: Sequence alignment shows that Emit3 and Tfiibl1 have a conserved zinc-ribbon motif. Tthe: Tetrahymena thermophila ; Tmal: Tetrahymena malaccensis ; Tell: Tetrahymena elliotti ; Tbor: Tetrahymena borealis ; Tvor: Tetrahymena vorax ; Tsp: Tetrahymena sp .; Tepi: Tetrahymena empidokyrea ; Tsha: Tetrahymena shanghaiensis ; Tpar: Tetrahymena paravorax ; Hasp: Homo sapiens ; Atha: Arabidopsis thaliana ; Scer: Saccharomyces cerevisiae . Lower panel: The N-terminal region contains a putative zinc-ribbon motif, with the positions of the zinc-binding cysteines indicated. The zinc-ribbon structure of Emit3 is similar to that of Pyrococcus furiosus TFIIB (PfTFIIB), as retrieved from the Protein Data Bank (PDB code: 1pft). Homology modeling of the C-terminal regions is presented in . ( E ) DNA FISH analysis of REP2 IESs (magenta) in emit3 Δ and WT cells harvested at 32 h after the induction of conjugation. REP2 IESs are retained in progeny MACs of the mutant. Scale bars: 10 μm. ( F ) The scnRNA (indicated by an arrowhead) was detected in WT cells but not in emit3 Δ cells.

Article Snippet: The Xpb2 coding sequence was synthesized by the ThermoFisher GeneArt DNA synthesis service (Thermo Fisher Scientific, Waltham, MA).

Techniques: Activity Assay, Conjugation Assay, Synthesized, Activation Assay, Gene Expression, Sequencing, Binding Assay, Mutagenesis