YY1 and RTCB in mouse uterine decidualization and embryo implantation

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

In vitro selections with RNAs of variable length converge on a robust catalytic core

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.

MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

New Deoxyribozymes for the Native Ligation of RNA

Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3′-5′-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.