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

  • ️Fri Jan 01 2021

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

Barbora Mikolaskova et al. Int J Mol Sci. 2021.

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.

Keywords: Cka1; Nrl1; fission yeast; phosphorylation; pre-mRNA splicing; protein–protein interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Physical interactions between Nrl1 and selected RNA-processing factors analyzed by the BACTH assay. (a) In vivo protein–protein interaction studies of Nrl1 and RNA-processing factors Ctr1, Ntr2, Syf3, and Mtl1 helicase. The nrl1, ctr1, mtl1, ntr2, and syf3 were sub-cloned into the pUT18C and pKT25 plasmids. E. coli cells transformed with the indicated constructs were plated on standard LB agar plates and LB agar plates containing X-gal and IPTG. The pUT18-zip and pKT25-zip plasmids and interactions between the Ctr1 and Mtl1 were used as positive controls. As negative controls, the empty vectors pUT18C and pKT25 were used. (b) Intensities of interactions between the studied proteins were evaluated by β-Galactosidase activity assay. Quantification of β-galactosidase activities of transformants are shown in Miller units. Data represent mean values ± S.D. from eight independent biological replicates.

Figure 2
Figure 2

Schematic representation of known Nrl1 domains and motifs and the regions that were truncated.

Figure 3
Figure 3

Physical interactions between wild-type Nrl1, truncated Nrl1 proteins, and RNA-processing factors Ctr1, Mtl1, Ntr2, and Syf3 analyzed by BACTH assay. The interaction between the Ctr1 and Mtl1 was used as a positive control. Data represent mean values ± S.D. from eight independent biological replicates. Statistical significance of truncated domain construct interactions compared to Nrl1 and selected RNA-processing factor interactions was determined using one-way analysis of variance (ANOVA) (p-values: *–p ≤ 0.05 and **–p ≤ 0.01).

Figure 5
Figure 5

Fragmentation (MS/MS) spectra of newly identified Nrl1 phosphopeptides. Annotated fragmentation spectrum of a singly phosphorylated Nrl1 peptide (SSpSSVGSAPLLR) containing pS86 and a singly phosphorylated Nrl1 peptide (pSLEIKYDEER) containing pS122. Overall fragment coverage is indicated in the peptide sequences below the spectra with the b-ions in blue and y-ions in red. The asterisks in the spectra (*) denote peptide fragments with a neutral loss of H3O4P (97.98 Da) originating from the corresponding phosphorylated fragment ions.

Figure 6
Figure 6

Expression, purification, and in vitro phosphorylation of Nrl1 fragment by Cka1 protein kinase. (a) Nrl1 protein fragment (1–268aa) was expressed in fusion with maltose binding protein (MBP) in E. coli. (b) MBP-Nrl1(1–268aa) protein was affinity purified using amylose resin. As a control of purification efficiency, MBP was used. (c) MBP-Nrl1(1–268aa) purified from E. coli was incubated with Cka1-TAP purified from S. pombe cells and analyzed by mass spectrometry. Two Nrl1 serine residues that were found to be phosphorylated are marked by asterisks and highlighted in red.

Figure 7
Figure 7

Analysis of gene expression in nrl1Δ and phosphorylation-site mutants nrl1S/A and nrl1S/D. RNA was isolated from cells in the exponential phase (OD600 = 0.5–0.6), and gene expression of SPBPB2B2.01, puf5, SPBC23G7.10c, gcd1, ght1, and agl1 was analyzed using qPCR. The data represent mean values ± S.D. of transcript levels relative to wild-type nrl1+ after normalization to act1 from four independent biological replicates. Statistical significance in expression of studied genes in nrl1 mutants compared to wild-type nrl1+ was determined using one-way analysis of variance using ANOVA (p-values: *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001).

Figure 8
Figure 8

Analysis of splicing efficiency in nrl1Δ and phosphorylation-site mutants nrl1S/A and nrl1S/D. The relative splice-index (SI relative) is shown for the 3rd intron of mug161 and 1st introns of SPBC557.05, SPBC1604.04, caf16, cbc3, and mbx1. The data represent mean values ± S.D. of splicing index (SI) relative to wild-type from three independent biological replicates. Statistical significance in splicing of studied genes in nrl1 mutants compared to wild-type nrl1+ was determined using one-way analysis of variance (ANOVA) (p-values: *–p ≤ 0.05 and ***–p ≤ 0.001).

Figure 9
Figure 9

Schematic representation of the identified Nrl1 phosphorylation and domain-specific interactions of Nrl1 protein with various RNA-processing factors.

Figure 10
Figure 10

Scheme illustrating the principle of the BACTH assay. (a) Proteins T25-X and T18-Z that do not interact will not reconstitute adenylate cyclase activity. (b) Proteins T25-X and T18-Z that interact will reconstitute adenylate cyclase activity leading to production of cAMP. (c) Cyclic adenosine monophosphate (cAMP) bound to catabolite activator protein (CAP) positively regulates β-galactosidase expression.

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