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.
Highlights
Eukaryotic cells use various mechanisms to regulate gene expression
This highly orchestrated process is driven by a multimegadalton ribonucleoprotein complex known as a spliceosome, comprising of five main small nuclear ribonucleoproteins, U1, U2, U4/U6, and U5, that associate with its cognate small nuclear RNA, and of numerous splicing proteins and associated factors [1,2,3,4]
Defining the protein domains that mediate protein–protein interactions can help to reveal the molecular function of proteins within their protein complexes
Summary
Eukaryotic cells use various mechanisms to regulate gene expression Among these processes, pre-mRNA splicing plays an important role. Pre-mRNA splicing is known as a process in which introns are removed from a pre-mRNA to create a mature RNA molecule This highly orchestrated process is driven by a multimegadalton ribonucleoprotein complex known as a spliceosome, comprising of five main small nuclear ribonucleoproteins (snRNP), U1, U2, U4/U6, and U5, that associate with its cognate small nuclear RNA (snRNA), and of numerous splicing proteins and associated factors [1,2,3,4]. It is well known that during the splicing events the spliceosome dynamically changes its composition and structure Since these changes are highly intricate and fine-tuned, they are susceptible to many alterations. Mutations of splicing factors or alterations in the mechanisms regulating the splicing processes might lead to tumorigenesis or various developmental diseases [5,6,7,8,9]
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