Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning.
Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning.
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DOI：
10.1016/j.ydbio.2021.03.021
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发表时间：
2021-08
影响因子：
2.7
通讯作者：
Macdonald PM
中科院分区：
文献类型：
--
作者：
Kenny A;Morgan MB;Macdonald PM
奥斯卡果蝇（osk） mRNA具有编码和非编码功能，后者需要通过卵发生进展。非编码活动由osk3 ' UTR介导。三种类型的顺式元件最直接地起作用，它们聚集在3 ' UTR的最后约120个核苷酸中：Bru1蛋白的多个结合位点，一个短的高度保守区域，以及靠近聚(a)尾部的富含a的序列。在这里，我们扩展了这些元件及其功能的表征，为osk非编码RNA功能和顺式元件的组成提供了新的见解。我们发现所有这三个元素都是微管组织中心（MTOC）正确定位所必需的，这是以前没有报道过的任何风险突变体的缺陷。通常情况下，在卵形成前阶段，MTOC位于卵母细胞的后部，这种分布是许多mrna从护理细胞转运到卵母细胞的强烈后部富集的基础。当osk非编码功能被破坏时，MTOC分散在卵母细胞中，osk mRNA在后部无法富集，但向卵母细胞的转运不受影响。先前的研究没有发现某些缺乏非编码活性的风险突变体的后侧富集缺失。这种差异可能是由于成像的目的是监测转运到卵母细胞，而不是后部富集。参与MTOC定位表明，osk的非编码功能可能与丢失具有类似影响的基因一起起作用，并且osk功能可能扩展到需要这些基因的其他过程中。对osk非编码功能所需的顺式元件的进一步表征包括完成最高度保守区域的饱和诱变，为评估候选结合因子的可能贡献提供了关键信息。最靠近3 '的顺式元素是一组富含a的序列，即ARS。ARS和poly(A) tail的紧密并列和结构相似性提高了它们包含一个扩展的A-富元素的可能性，这是ask非编码功能所必需的。我们发现，多(A)尾的缺失不会模仿ARS突变的影响，既不会导致卵子发生阻滞，也不会导致胚胎发生前阶段卵母细胞中osk mRNA的错误定位。因此，ARS和聚(A)尾对于ask非编码RNA的功能是不可互换的，这表明ARS的作用不在于募集聚(A)结合蛋白（PABP），即与聚(A)尾结合的蛋白。此外，尽管PABP与osk mRNA从哺乳细胞转运到卵母细胞有关，但ARS突变与聚(A)尾缺失并没有破坏osk mRNA进入卵母细胞的转运。我们得出结论，PABP间接作用于osk mRNA的转运，或者与osk mRNA相关，不依赖于富含a的结合位点。尽管多聚(A)尾不是osk mRNA转运到卵母细胞所必需的，但它的缺失与卵子形成后期新的osk mRNA定位缺陷有关，可能揭示了定位过程中以前未被识别的步骤。
Drosophila oskar (osk) mRNA has both coding and noncoding functions, with the latter required for progression through oogenesis. Noncoding activity is mediated by the osk 3’ UTR. Three types of cis elements act most directly and are clustered within the final ~120 nucleotides of the 3’ UTR: multiple binding sites for the Bru1 protein, a short highly conserved region, and A-rich sequences abutting the poly(A) tail. Here we extend the characterization of these elements and their functions, providing new insights into osk noncoding RNA function and the makeup of the cis elements. We show that all three elements are required for correct positioning of the microtubule organizing center (MTOC), a defect not previously reported for any osk mutant. Normally, the MTOC is located at the posterior of the oocyte during previtellogenic stages of oogenesis, and this distribution underlies the strong posterior enrichment of many mRNAs transported into the oocyte from the nurse cells. When osk noncoding function was disrupted the MTOC was dispersed in the oocyte and osk mRNA failed to be enriched at the posterior, although transport to the oocyte was not affected. A previous study did not detect loss of posterior enrichment for certain osk mutants lacking noncoding activity. This discrepancy may be due to use of imaging aimed at monitoring transport to the oocyte rather than posterior enrichment. Involvement in MTOC positioning suggests that the osk noncoding function may act in conjunction with genes whose loss has similar effects, and that osk function may extend to other processes requiring those genes. Further characterization of the cis elements required for osk noncoding function included completion of saturation mutagenesis of the most highly conserved region, providing critical information for evaluating the possible contribution of candidate binding factors. The 3’-most cis element is a cluster of A-rich sequences, the ARS. The close juxtaposition and structural similarity of the ARS and poly(A) tail raised the possibility that they comprise an extended A-rich element required for osk noncoding function. We found that absence of the poly(A) tail did not mimic the effects of mutation of the ARS, causing neither arrest of oogenesis nor mispositioning of osk mRNA in previtellogenic stage oocytes. Thus, the ARS and the poly(A) tail are not interchangeable for osk noncoding RNA function, suggesting that the role of the ARS is not in recruitment of Poly(A) binding protein (PABP), the protein that binds the poly(A) tail. Furthermore, although PABP has been implicated in transport of osk mRNA from the nurse cells to the oocyte, mutation of the ARS in combination with loss of the poly(A) tail did not disrupt transport of osk mRNA into the oocyte. We conclude that PABP acts indirectly in osk mRNA transport, or is associated with osk mRNA independent of an A-rich binding site. Although the poly(A) tail was not required for osk mRNA transport into the oocyte, its absence was associated with a novel osk mRNA localization defect later in oogenesis, potentially revealing a previously unrecognized step in the localization process.
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DOI：
10.1261/rna.048298.114
发表时间：
2015-06
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Kanke M;Jambor H;Reich J;Marches B;Gstir R;Ryu YH;Ephrussi A;Macdonald PM
通讯作者：
Macdonald PM
影响因子：
64.5
作者：
LEHMANN, R;NUSSLEINVOLHARD, C
通讯作者：
NUSSLEINVOLHARD, C
影响因子：
4.5
作者：
Kim G;Pai CI;Sato K;Person MD;Nakamura A;Macdonald PM
通讯作者：
Macdonald PM
影响因子：
56.9
作者：
Brendza, RP;Serbus, LR;Saxton, WM
通讯作者：
Saxton, WM
影响因子：
9.2
作者：
Glotzer, JB;Saffrich, R;Ephrussi, A
通讯作者：
Ephrussi, A