Expression of zebrafish cpsf6 in embryogenesis and role of protein domains on subcellular localization

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Abstract

CPSF6 is a component of the CFIm complex, involved in mRNA 3′end processing. Despite increasing interest on this protein as a consequence of proposed roles in cancer and HIV infection, several aspects of CPSF6 biological function are poorly understood. In this work we studied the expression of the zebrafish ortholog cpsf6 in early stages of embryo development. Quantitative RT-PCR studies showed that zebrafish cpsf6 mRNA is maternally inherited and that its concentration markedly decreases during early development. We found a generalized distribution of cpsf6 mRNA in early stages through whole mount hybridization experiments. By performing Western blot, we also found a decrease in zebrafish Cpsf6 levels during development. Our analysis of the subcellular localization of this protein using a heterologous system showed a distinct pattern characterized by the presence of nuclear foci. We also studied the relevance of different protein domains on subcellular localization, showing that the C-terminal domain is critical for nuclear localization. Collectively, our results showed that cpsf6 expression changes during early development and that the subcellular localization of the protein is similar to that of the human ortholog.

Introduction

Cleavage and Polyadenylation Factor 6 (CPSF6) is a component of the complex machinery that regulates pre-mRNA polyadenylation. The initial steps of this postranscriptional process requires the action of three multiprotein complexes, CFIm, CFIIm and CstF, which constitute the Cleavage and Polyadenylation Complex (CPA) (reviewed in (Tian and Manley, 2016)). CFIm is a heterotetramer containing a dimer of CPSF5 in combination with a dimer of either CPSF6 or CPSF7 (Rüegsegger et al., 1998, 1996).

CPSF6 shows three domains defined basing on structural homology. The N-terminal domain contains an RNA Recognition Motif (RRM). The central Proline-rich domain (PRD) is followed by an RS-like C-terminal domain (RSLD), enriched in Arg-Asp/Glu repeats. Despite the presence of an RRM motif, the interaction with polyadenylation sequences on target mRNA is mediated by CPSF5 (Tian and Manley, 2016). Nevertheless, CPSF6 regulates the specificity of the complex, as suggested by evidence showing that its depletion enhanced the shortening of mRNAs by favoring the use of proximal polyadenylation sites in 3’ untranslated regions (UTRs) (Gruber et al., 2012; Martin et al., 2012). Evidence from primordial germ cells in mice (Sartini et al., 2008) and Medaka (Sasado et al., 2017) also support a role of CPSF6 in alternative cleavage and polyadenylation.

CPSF6 has also been related to pathological processes. For example, a role in Acquired Immunodeficiency Syndrome (AIDS) was proposed, since binding of CPSF6 to HIV capsid proteins was shown to be required for efficient nuclear import (Lee et al., 2010). Accordingly, CPSF6 depletion reduced HIV infectivity in macrophages (Bejarano et al., 2019). Similarly, a C-terminally truncated form of CPSF6 was able to reduce infection in HEK-293 and MOLT-4 human T-cell lines (Hori et al., 2013). In addition, independent evidence has suggested that CPSF6 may cooperate with oncogenic mechanisms. We have shown that CPSF6 expression is enhanced by p53 point mutants (Girardini et al., 2011), frequently found in human cancers and that its depletion reduces migration in vitro. We also found that CPSF6 is part of a gene signature that correlates with reduced survival in breast cancer. Similarly, CPSF6 depletion reduced in vitro proliferation of aggressive breast cancer cell lines and tumor formation in a xenograft model (Binothman et al., 2017). Additional evidence also reinforces the notion that CPSF6 may play a role in cancer. CPSF6 fusions to other genes have been described in some patients with hematological malignancies (Hidalgo-Curtis et al., 2008; Qin et al., 2018). Moreover, forced expression of the epithelial-to-mesenchymal transition promoter factor SNAIL, in colon cancer cells, increased CPSF6 protein levels (Larriba et al., 2010). Collectively, this evidence suggests a complex role for CPSF6.

In order to expand our knowledge on CPSF6 biological role, we studied the expression of its zebrafish ortholog during early embryonic development. According to our results cpsf6 mRNA is present in 1–2 cell stage. We found a marked decrease in cpsf6 mRNA and protein levels at later stages. The analysis of spatial distribution of cpsf6 mRNA suggests a ubiquitous expression in the early embryo. By expressing zebrafish Cpsf6 fused to GFP in mammalian cells we showed that the protein is present in nuclear paraspeckles. Moreover, we studied the relevance of structural domains on subcellular localization. We found that the RSLD is required for nuclear localization. Our work provides the basis for the use of zebrafish embryos as a model system to study Cpsf6 function.

Section snippets

Sequence analysis of CPSF6 proteins

In order to isolate a sequence coding for zebrafish Cpsf6, we performed RT-PCR on total RNA extracted from 1 to 2 cell stage embryos. We obtained a fragment of the expected size that was cloned to generate pGcpsf6 plasmid and sequenced. The protein sequence deduced from the amplified fragment was compared with sequences from vertebrate orthologs (Fig. 1a). We observed high percent identity values, exceeding 80% (Fig. 1b). Among the sequences examined, zebrafish Cpsf6 shared the highest identity

Discussion

The involvement of CPSF6 in pathological processes is attracting increasing attention; however, the mechanisms underlying its pathologic effects are not well understood. CPSF6 plays a role in the initial steps of 3’ end mRNA polyadenylation. Moreover, independent evidence has documented that CPSF6 regulates the use of alternative polyadenylation sites (Sartini et al., 2008; Sasado et al., 2017). Nevertheless, the mechanisms activated by CPSF6 in pathological conditions have not been related to

Zebrafish embryos maintenance

Adult zebrafish (Danio rerio, strain AB) were maintained at 28 °C on a 14 h light/10 h dark cycle as previously described (Westerfield, 2007). Embryos were staged according to development at 28 °C (Kimmel et al., 1995), and handled in compliance with international guidelines. Protocols were approved by the Research Bioethics Comittee, Facultad de Ciencias Bioquímicas y Farmacéuticas - Universidad Nacional de Rosario.

RNA extraction and reverse transcription

Total RNA was isolated from zebrafish embryos using TRIZOL (Invitrogen)

Declaration of competing interest

None.

Acknowledgements

We are grateful to Sebastian Graziati for zebrafish husbandry, to Rodrigo Vena and Enrique Morales for technical assistance with confocal microscopy. We also thank Cecilia Larocca for assistance with image analysis. This work was supported by grants to Javier Girardini from CONICET (PIP 114 201001 00141) and from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT. PICT 1221).

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