Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry

Abstract

Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51–110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein–protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.

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

“…Prp43 activity contributes to the maintenance of spliceosome integrity because reduced Prp43 function promotes the use of structurally aberrant spliceosomes and the splicing of suboptimal premRNA substrates (Pandit et al 2006;Koodathingal et al 2010;Mayas et al 2010;Chen et al 2013). In addition to features of the postcatalytic spliceosome, specific changes in spliceosome composition linked to ATP hydrolysis by the Prp2, Prp16, and Prp22 DExD/H-box proteins render defective splicing complexes sensitive to Prp43 recruitment and ATP-dependent dissociation (Chen et al 2013).Data from several groups implicate three Prp43-interacting factors in the regulation of this protein's role in pre-mRNA splicing (Spp382/Ntr1) and pre-rRNA processing (Sqs1/Pfa1 and Pxr1/Gno1) Lebaron et al 2005;Tsai et al 2005;Boon et al 2006;Pandit et al 2006;Tanaka et al 2007;Tsai et al 2007;Lebaron et al 2009;Pertschy et al 2009;Walbott et al 2010;Christian et al 2014). Spp382 is an essential pre-mRNA splicing factor required for Prp43 recruitment to the spliceosome.…”

“…Data from several groups implicate three Prp43-interacting factors in the regulation of this protein's role in pre-mRNA splicing (Spp382/Ntr1) and pre-rRNA processing (Sqs1/Pfa1 and Pxr1/Gno1) Lebaron et al 2005;Tsai et al 2005;Boon et al 2006;Pandit et al 2006;Tanaka et al 2007;Tsai et al 2007; Lebaron et al 2009;Pertschy et al 2009;Walbott et al 2010;Christian et al 2014). Spp382 is an essential pre-mRNA splicing factor required for Prp43 recruitment to the spliceosome.…”

“…Point mutations within the SPP382, PXR1, and SQS1 G-patch coding segments can block or weaken the corresponding protein interaction with Prp43 and inhibit pre-mRNA splicing or rRNA processing Pandit et al 2006;Tanaka et al 2007;Lebaron et al 2009). In addition, Spp382 and Sqs1 peptides bearing the G-patch domain stimulate the RNA-dependent helicase activity of Prp43 in vitro (Tanaka et al 2007;Lebaron et al 2009;Christian et al 2014). While critical to each protein's biological activity, prior work leaves unresolved whether the Spp382, Sqs1, and Pxr1 G-patch domains are equivalent interfaces that promote a common Prp43 activity or serve a more complex function in the pre-mRNA splicing and rRNA processing pathways.…”

Structure and target interaction of a G-quadruplex RNA-aptamer

“…Prior to protease digestion, hIL-6R was covalently cross-linked to AIR-3A by means of UVirradiation. [36][37][38] Purified samples of this covalent adduct, termed herein as hIL-6R: AIR-3A, were subjected to a standard trypsin digestion protocol and the final peptides were analyzed by LC-MS. Samples of hIL-6R UV-irradiated in the absence of AIR-3A served as a reference.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…In recent years, chemical protein-protein cross-linking and UV-induced protein-nucleic acid cross-linking, in combination with mass spectrometry, have emerged as complementary methods for obtaining information about the spatial arrangement of proteins in complexes and in RNPs [9,10]. In the case of UV-induced protein-RNA cross-linking, MS has been applied to identify the cross-linked proteins by standard quantitative MS-based proteomic approaches [11][12][13].…”