Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland;
Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland.
Proc Natl Acad Sci U S A. 2019 Feb 19;116(8):2935-2944. doi: 10.1073/pnas.1808696116. Epub 2019 Feb 4.
Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.
人抗原 R（HuR）是含有腺苷酸/尿苷酸丰富元件（AREs）的细胞 mRNA 的关键调节剂。这些是 3'非翻译区中主要的一类元件，靶向这些 mRNA 进行快速降解。HuR 包含三个 RNA 识别基序（RRMs）：串联的 RRM1 和 2，后面跟着一个柔性接头和 C 末端的 RRM3。虽然 RRM1 和 2 的结构已得到表征，但对 RRM3 知之甚少。在这里，我们展示了与不同 ARE 基序结合的 RRM3 的 1.9 Å 分辨率晶体结构。该结构以及生物物理方法和细胞培养测定揭示了 RRM3 ARE 识别和二聚化的机制。虽然可以结合多个 RNA 基序，但通过结合两个寄存器，可以识别典型的 AUUUA 五聚体基序。此外，RRM3 形成同源二聚体以增加其 RNA 结合亲和力。最后，尽管 HuR 稳定含有 ARE 的 RNA，但我们发现 RRM3 会产生相反的效果，如在基于细胞的 ARE 报告基因测定和含有多个 AUUUA 基序的天然 HuR mRNA 靶标上的 qPCR 所示，可能通过与 RRM12 竞争来实现。
