Hou Linlin, Klug Gabriele, Evguenieva-Hackenberg Elena
Institute of Microbiology and Molecular Biology, Heinrich-Buff-Ring 26-32, D-35392 Gießen, Germany.
Institute of Microbiology and Molecular Biology, Heinrich-Buff-Ring 26-32, D-35392 Gießen, Germany
Nucleic Acids Res. 2014 Nov 10;42(20):12691-706. doi: 10.1093/nar/gku969. Epub 2014 Oct 17.
The archaeal exosome is a phosphorolytic 3'-5' exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation.
古菌外切体是一种磷酸解3'-5'外切核糖核酸酶复合体。在反向反应中，它能合成富含A的RNA尾巴。其RNA结合帽包含真核生物直系同源物Rrp4和Csl4，以及一个注释为DnaG的古菌特异性亚基。在嗜热栖热菌中，DnaG和Rrp4而非Csl4对聚（rA）表现出偏好。古菌DnaG在TOPRIM结构域两侧含有功能未知的N端和C端结构域（NTD和CTD）。我们发现，NT和TOPRIM结构域在所有古菌中具有相当高的保守性，而CTD的保守性与外切体的存在相关。我们表明，NTD是一个新的RNA结合结构域，具有聚（rA）偏好，在RNA结合中与TOPRIM结构域协同作用。一致地，一种包含全长Csl4和DnaG的NTD的融合蛋白导致外切体对富含A的RNA的降解增强。我们还发现，DnaG能强烈结合天然和体外转录的rRNA，并使其通过外切体进行多聚核苷酸化。此外，具有杂聚尾巴的rRNA衍生转录本比其无尾变体被外切体降解得更快。基于我们的数据，我们提出古菌DnaG是一种RNA结合蛋白，在与外切体相关的情况下，它参与稳定RNA的靶向降解。
