Department of Biochemistry and Molecular Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, United States of America.
PLoS One. 2022 Jul 29;17(7):e0271453. doi: 10.1371/journal.pone.0271453. eCollection 2022.
Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3' UTR. Aside from the SECIS, selenoprotein mRNA 3' UTRs are not conserved between different selenoproteins within a species. In contrast, the 3'-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3'-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3' UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.
硒蛋白含有第 21 种氨基酸——硒代半胱氨酸(Sec),当存在特殊发夹序列 SECIS 元件时,Sec 会在特定的 UGA 密码子处掺入。除了 SECIS 元件之外,硒蛋白 mRNA 的 3'UTR 在物种内的不同硒蛋白之间没有保守性。相比之下,给定硒蛋白的 3'-UTR 在物种之间通常是保守的,这支持了 3'-UTR 中非 SECIS 的顺式作用元件对硒蛋白表达进行转录后调控的假说。为了确定 SECIS 背景中的一个这样的顺式作用元件的功能,我们选择专注于血浆硒蛋白 SELENOP,它作为一种含有 10 个 Sec 残基的硒转运蛋白,是维持硒稳态所必需的。它是独特的,因为它的 mRNA 在高度保守的 843 个核苷酸 3'UTR 中包含两个 SECIS 元件。在这里,我们使用 RNA 亲和层析鉴定出 PTBP1 是与两个 SECIS 元件之间的序列特异性相互作用的主要 RNA 结合蛋白。然后,我们使用 CRISPR/Cas9 基因组编辑删除了第一个 SECIS 元件周围的两个区域。我们发现这些序列参与调节 SELENOP mRNA 和蛋白水平,这些水平随硒浓度的变化而反向改变。