A sulfotransferase gene BnSOT-like1 has a minor genetic effect on seed glucosinolate content in Brassica napus

The theory and associated selection methods of classical quantitative genetics are based on the multifactorial or polygene hypothesis.Major genes or quantitative trait loci(QTL)in modern quantitative genetics based on a“major gene plus polygenes”genetic system have been paid much attention in genetic studies.However,it remains unclear how the numerous minor genes act,although the polygene theory has sustained genetic improvement in plants and animals for more than a hundred years.In the present study,we identified a novel minor gene,BnSOT-like1(BnaA09g53490D),which is a sulfotransferase(SOT)gene catalyzing the formation of the core glucosinolate(GSL)structure in Brassica napus.This gene has been occasionally found during investigations of plant height-related genes,but has not been identified by QTL mapping because of its small phenotypic effects on GSL content.The overexpression of BnSOT-like1 up-regulated the expression of aliphatic GSL-associated genes,leading to a high seed aliphatic GSL content,and the overexpression of the allelic gene Bnsot-like1 did not increase seed GSL content.These findings suggest that the SOT gene has a marked effect on a quantitative trait from a reverse genetics standpoint,but a minor effect on the quantitative trait in its natural biological state.Because of the redundancy of GSL biosynthetic genes in the allotetraploid species B.napus,mutations of a single functional gene in the pathway will not result in significant phenotypic changes,and that the genes in biosynthetic pathways such as BnSOT-like1 in our study have minor effects and may be called polygenes in contrast to the reported three regulatory genes(BnHAG1s)which strongly affect GSL content in B.napus.The present study has shed light on a minor gene for a quantitative trait.

Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit

Many long noncoding RNAs(lncRNAs)have been identified through siRNA-based screening as essential regulators of embryonic stem cell(ESC)pluripotency.However,the biological and molecular functions of most lncRNAs remain unclear.Here,we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs.Unexpectedly,all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination.Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity.We further showed that several small hairpin RNAs(shRNAs)previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs,indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs.Interestingly,linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body(EB)differentiation.By reintroducing RNA products generated from the linc1343 locus,we found that two snoRNAs,Snora73a and Snora73b,but not lncRNAs,could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs.Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture;however,we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

哺乳动物TRIM71蛋白RNA结合特性的分子机制研究

TRIM71 is an RNA-binding protein with ubiquitin ligase activity.Numerous functions of mammalian TRIM71,including cell cycle regulation,embryonic stem cell(ESC)self-renewal,and reprogramming of pluripotent stem cells,are related to its RNA-binding property.We previously reported that a long noncoding RNA(lnc RNA)Trincr1 interacts with mouse TRIM71(m TRIM71)to repress FGF/ERK pathway in mouse ESCs(m ESCs).Herein,we identify an RNA motif specifically recognized by m TRIM71 from Trincr1 RNA,and solve the crystal structure of the NHL domain of m TRIM71 complexed with the RNA motif.Similar to the zebrafish TRIM71,m TRIM71 binds to a stem-loop structured RNA fragment of Trincr1,and an adenosine base at the loop region is crucial for the m TRIM71 interaction.We map similar hairpin RNAs preferably bound by TRIM71 in the m RNA UTRs of the cell-cycle related genes regulated by TRIM71.Furthermore,we identify key residues of m TRIM71,conserved among mammalian TRIM71 proteins,required for the RNA-binding property.Single-site mutations of these residues significantly impair the binding of TRIM71 to hairpin RNAs in vitro and to m RNAs of Cdkn1a/p21 and Rbl2/p130 in m ESCs.Furthermore,congenital hydrocephalus(CH)specific mutation of m TRIM71 impair its binding to the RNA targets as well.These results reveal molecular mechanism behind the recognition of RNA by mammalian TRIM71 and provide insights into TRIM71 related diseases.

A long non-coding RNA LncSync regulates mouse cardiomyocyte homeostasis and cardiac hypertrophy through coordination of miRNA actions

Dear Editor,Maintenance of cardiomyocyte(CM)homeostasis is essential for normal heart function.Long-term imbalance in heart homeostasis could elicit irreversible adaptive change in cell structure and function and tissue architecture,exemplified as cardiac hyper-trophy and fibrosis,and eventually develop into heart failure(Shiojima et al.,2005).Therefore,identifying new genes and path-ways regulating CM homeostasis may help better understand the cause of cardiac hypertrophy.

Large Noncoding RNAs Are Promising Regulators in Embryonic Stem Cells

Embryonic stem cells （ESCs） hold great promises for treating and studying numerous devastating diseases. The molecular basis of their potential is not completely understood. Large noncoding RNAs （lncRNAs） are an important class of gene regulators that play essential roles in a variety of physiologic and pathologic processes. Dozens of lncRNAs are now identified to control ESC self-renewal and differentiation. Research on lncRNAs may provide novel insights into manipulating the cell fate or reprogramming somatic cells into induced pluripotent stem cells （iPSCs）. In this review, we summarize the recent research efforts in identifying functional lncRNAs and understanding how they act in ESCs, and discuss various future directions of this field.

KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2

The pluripotent state of embryonic stem cells(ESCs)is regulated by a sophisticated network of transcription factors.High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs(h ESCs).However,the functional role of KLF17 in naive state is not clear.Here,by employing various gain and loss-of-function approaches,we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in h ESCs.Mechanistically,we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17.Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency.Furthermore,we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species.Finally,we show that Klf17 may be essential for early embryo development in mouse.These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

Dgcr8 deletion in the primitive heart uncovered novel microRNA regulating the balance of cardiac-vascular gene program

Primitive mammalian heart transforms from a single tube to a four-chambered muscular organ during a short developmental window.We found that knocking out global microRNA by deleting Dgcr8 microprocessor in Mespl cardiovascular progenitor cells lead to the formation of extremely dilated and enlarged heart due to defective cardiomyocyte(CM)differentiation.Transcriptome analysis revealed unusual upregulation of vascular gene expression in Dgcr8 cKO hearts.Single cell RNA sequencing study further confirmed the increase of angiogenesis genes in single Dgcr8 cKO CM.We also performed global microRNA profiling of E9.5 heart for the first time,and identified that miR-541 was transiently highly expressed in E9.5 hearts.Interestingly,introducing miR-541 back into microRNA-free CMs partially rescued their defects,downregulated angiogenesis genes and significantly upregulated cardiac genes.Moreover,miR-541 can target Ctgf and inhibit endothelial function.Our results suggest that micro-RNAs are required to suppress abnormal angiogenesis gene program to maintain CM differentiation.

Acidic pH transiently prevents the silencing of self-renewal and dampens microRNA function in embryonic stem cells

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells.However,little is known about its regulatory roles in gene expression and cell fate determination.Here,we confirm that glycolytic metabolism and lactate production decrease during the differentiation of mouse embryonic stem cells(mESCs).Importantly,acidic pH due to lactate accumulation can transiently prevent the silencing of mESC self-renewal in differentiation conditions.Furthermore,acidic pH partially blocks the differentiation of human ESCs(hESCs).Mechanistically,acidic pH downregulates AGO1 protein and de-represses a subset of mRNA targets of miR-290/302 family of microRNAs which facilitate the exit of naive pluripotency state in mESCs.Interestingly,AGO1 protein is also downregulated by acidic pH in cancer cells.Altogether,this study provides insights into the potential function and underlying mechanism of acidic pH in pluripotent stem cells(PSCs).

Significant differences of function and expression of microRNAs between ground state and serum-cultured pluripotent stem cells

Serum- and 2i-cultured embryonic stem cells （ESCs） show different epigenetic landscapes and tran- scriptomic profiles. The difference in the function and expression of microRNAs （miRNAs） between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with 〉100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8-/- or Dicerl-I- but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8/- ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum- and 2i- cultured ESCs.