Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae
Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae
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DOI：
10.1534/genetics.115.176461
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发表时间：
2015-05-01
期刊：
影响因子：
3.3
通讯作者：
Rymond, Brian C.
中科院分区：
文献类型：
--
作者：
Banerjee, Daipayan;McDaniel, Peter M.;Rymond, Brian C.
Prp 43 DExD/H-box蛋白是生物化学上不同的前信使RNA和核糖体RNA（rRNA）成熟途径进展所必需的。在酿酒酵母中，Spp 382/Ntr 1、Sqs 1/Pfa 1和Pxr 1/Gno 1蛋白质被认为是在剪接体解离（Spp 382）和rRNA加工（Sqs 1和Pxr 1）期间Prp 43解旋酶活化所必需的辅因子。虽然在一级序列上不同，但这些Prp 43结合蛋白各自含有功能所需的短的富含甘氨酸的G-补丁基序，并被认为在蛋白质或核酸识别中起作用。在这里，酵母双杂交，结构域交换，和定点诱变方法被用来调查G-补丁域的活性和可移植性。我们的研究结果表明，Spp 382，Sqs 1，和PXR 1 G-补丁不同Prp 43双杂交反应和重建Spp 382和PXR 1 RNA加工因子的能力。G-补丁蛋白重建没有相关的Prp 43双杂交反应的表观强度，这表明该域的功能超出了Prp 43系链。事实上，虽然对PXR 1活性至关重要，但PXR 1 G-补丁似乎对酵母双杂交相互作用贡献不大。相反，Pxr 1（氨基酸102-149）内的主要Prp 43结合位点的缺失不会阻碍rRNA加工，但会影响小核仁RNA（snoRNA）生物合成，导致选择snoRNA的轻微延伸形式的积累，这是prp 43功能丧失突变体意外共享的表型。这些和相关的观察揭示了Spp 382，Sqs 1和Pxr 1蛋白如何与Prp 43相互作用的差异，并提供了将G-补丁身份与途径特异性DExD/H-盒解旋酶活性联系起来的证据。
The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102-149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.