Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

Abstract

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe

“…Recently, by searching for novel spliceosome-associated factors in the fission yeast S. pombe , we identified Nrl1 protein as a factor that is required not only for proper pre-mRNA splicing of a subset of genes and non-coding RNAs, but also for the maintenance of genome stability by both suppressing R-loops and promoting HR repair. Interestingly, among its top interactors, we have identified the G-patch domain-containing protein Gpl1 [ 41 , 43 ]. It has also been shown that the gpl1 Δ mutant has canonical splicing defects with broad increases in pre-mRNA species and decreases in mature mRNA species, which suggested that Gpl1 might be a novel factor implicated in the regulation of pre-mRNA splicing [ 44 ].…”

“…Yeast cell powders (40 g) were prepared from frozen cell pellets using SPEX SamplePrep 6770 Freezer/Mill (SPEX SamplePrep, Metuchen, NJ, USA) cooled by liquid nitrogen. Proteins were extracted using IPP150 buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF and complete protease and phosphatase inhibitors) in a ratio of 1 g of yeast cell powder to 3 mL of IPP150 buffer and affinity purified as described previously [ 43 , 64 ]. Briefly, 500 µL of IgG Sepharose™ 6 Fast Flow beads (GE Healthcare, Uppsala, Sweden) was equilibrated with IPP150 buffer, mixed with protein extract and incubated on rotatory wheel for 2 h at 4 °C.…”

“…Recently, we reported that the G-patch protein Gpl1 in the fission yeast S. pombe interacts with proteins involved in pre-mRNA splicing [ 41 , 42 , 43 ]. Previous studies also have shown that depletion of Gpl1 causes canonical splicing defects with broad increases in pre-mRNA species and decreases in mature mRNA species [ 44 ].…”

Adenovirus protein VII binds the A-box of HMGB1 to repress interferon responses

“…Protein-protein interaction between bait and prey reconstitutes adenylate cyclase, drives synthesis of cyclic-AMP, and leads to a red colony coloring on MacConkey agar plates (Fig 1A). This B2H system has previously been employed to quantitatively interrogate protein-protein interaction of various bacterial, fungal, and mammalian proteins [38][39][40][41]. We first sought to replicate our previous finding that protein VII interacts with HMGB1 [12] S1).…”

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…A main challenge in the study of signalling networks is to show the physiological context and significance of putative interactions. The BACTH system has been extensively used to demonstrate the specificity of protein interactions [67][68][69][70][71][72]. However, despite initial optimism [41,56,57], reports of using it to approach the regulation of protein complexes are difficult to find [73].…”