RNAi Screen of RING/U-Box Domain Ubiquitin Ligases Identifies Critical Regulators of Tissue Regeneration in Planarians.
RNAi Screen of RING/U-Box Domain Ubiquitin Ligases Identifies Critical Regulators of Tissue Regeneration in Planarians.
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DOI：
10.3389/fcell.2021.803419
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发表时间：
2021
影响因子：
5.5
通讯作者：
Zayas RM
中科院分区：
文献类型：
--
作者：
Allen JM;Balagtas M;Barajas E;Cano Macip C;Alvarez Zepeda S;Iberkleid I;Duncan EM;Zayas RM
再生过程依赖于信号的解释来协调细胞行为。已知泛素介导的信号传导在许多细胞和生物学背景中的作用是重要的，但其在再生中的作用还不清楚。为了研究泛素化如何影响体内组织再生，我们正在研究能够在几乎任何损伤后使用干细胞群体再生的真涡虫。在这里，我们使用RNAi来筛选在真涡虫干细胞和干细胞后代中高度表达的RING/U-box E3泛素连接酶。RNAi筛选鉴定出9个在再生中具有功能的基因，包括剪接体因子prpf 19和组蛋白修饰剂rnf 2;基于它们在发育过程中的已知作用，我们进一步研究了这两个基因。我们发现prpf 19是动物生存所必需的，但不是干细胞维持所必需的，这表明它在促进细胞分化中起作用。由于RNF 2是多梳抑制复合物1（PRC 1）的催化亚基，我们还研究了该复合物的其他推定成员（CBX和PHC）。我们在cbx和phc RNAi真涡虫中观察到一个显著的区域组织错配表型。为了鉴定PRC 1调控的基因，我们在敲低rnf 2或phc后进行了RNA-seq。尽管这些蛋白质被预测在相同的复合物中起作用，但我们发现在rnf 2与phc RNAi中差异表达的基因组在很大程度上是不重叠的。利用原位杂交，我们发现rnf 2调节组织类型内的基因表达水平，而phc对于基因表达的空间限制是必要的，研究结果与它们各自的体内表型一致。这项工作不仅揭示了RING/U-box E3连接酶在干细胞调控和再生中的作用，而且还确定了两个假定的PRC 1因子的差异基因靶点，这两个因子是维持涡虫细胞类型特异性基因表达所必需的。
Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling is known to be important in many cellular and biological contexts, but its role in regeneration is not well understood. To investigate how ubiquitylation impacts tissue regeneration in vivo, we are studying planarians that are capable of regenerating after nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor prpf19 and histone modifier rnf2; based on their known roles in developmental processes, we further investigated these two genes. We found that prpf19 was required for animal survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other putative members of this complex (CBX and PHC). We observed a striking phenotype of regional tissue misspecification in cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc. Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in rnf2 versus phc RNAi were largely non-overlapping. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression, findings consistent with their respective in vivo phenotypes. This work not only uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration, but also identified differential gene targets for two putative PRC1 factors required for maintaining cell-type-specific gene expression in planarians.
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影响因子：
14.9
作者：
Blum M;Chang HY;Chuguransky S;Grego T;Kandasaamy S;Mitchell A;Nuka G;Paysan-Lafosse T;Qureshi M;Raj S;Richardson L;Salazar GA;Williams L;Bork P;Bridge A;Gough J;Haft DH;Letunic I;Marchler-Bauer A;Mi H;Natale DA;Necci M;Orengo CA;Pandurangan AP;Rivoire C;Sigrist CJA;Sillitoe I;Thanki N;Thomas PD;Tosatto SCE;Wu CH;Bateman A;Finn RD
通讯作者：
Finn RD
影响因子：
5.3
作者：
BUNKER, CA;KINGSTON, RE
通讯作者：
KINGSTON, RE
DOI：
10.1007/978-1-4939-2303-8_7
发表时间：
2015-01-01
期刊：
IN SITU HYBRIDIZATION METHODS
影响因子：
--
作者：
Brown, David D. R.;Pearson, Bret J.
通讯作者：
Pearson, Bret J.
影响因子：
46.9
作者：
Bray, Nicolas L.;Pimentel, Harold;Pachter, Lior
通讯作者：
Pachter, Lior
影响因子：
64.5
作者：
CIECHANOVER, A;FINLEY, D;VARSHAVSKY, A
通讯作者：
VARSHAVSKY, A