Translational Biology, Medicine and Health Graduate Program, 1 Riverside Circle, Virginia Tech, Roanoke, VA 24016.
Biocomplexity Institute & Initiative, University of Virginia, 995 Research Park Blvd, Town Center III, 4th Floor, Charlottesville, VA 22911.
Hum Mol Genet. 2021 Jun 9;30(12):1101-1110. doi: 10.1093/hmg/ddab103.
The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.
导致普拉德-威利综合征（PWS）的最小基因组区域缺失了非编码 RNA SNORD116 簇；然而，SNORD116 的功能仍然是一个谜。该领域的先前工作揭示了一个诱人的可能性，即 NHLH2 基因的表达，该基因先前与肥胖和性腺功能减退有关，在 PWS 患者和分化的干细胞中下调。RNA:RNA 计算机建模鉴定了 SNORD116 和 NHLH2 mRNA 之间的几个潜在相互作用域。这些相互作用域之一在大多数检查的脊椎动物 NHLH2 mRNAs 中高度保守。包含 Nhlh2 mRNA 的构建体，包括其 3'-UTR，并与 c-myc 标记连接，在存在和不存在外源性表达的 Snord116 的情况下转染到下丘脑神经元细胞系中。在存在 Snord116 的情况下，Nhlh2 mRNA 的表达上调，这取决于构建体上使用的 3'UTR 的长度和类型。此外，使用放线菌素 D 阻止 N29/2 细胞中的新转录表明，上调是通过 Nhlh2 mRNA 在转录后立即的 45 分钟内增加稳定性来实现的。计算机建模还揭示，NHLH2 mRNA 中的单个核苷酸变异（SNV）可以降低 NHLH2:SNORD116 二联体的预测相互作用强度。事实上，使用包含该 SNV 的 Nhlh2 mRNA 构建体显着降低了 Snord116 增加 Nhlh2 mRNA 水平的能力。这些数据首次确定了 SNORD116 介导的 NHLH2 调节的基序和机制，阐明了缺失 SNORD116 snoRNAs 基因座导致 PWS 表型的机制。
