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Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve Mirna Biogenesis

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Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve Mirna Biogenesis | INVESTIGATION Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve miRNA Biogenesis Sri Ramulu N. Pullagura,*,† Bill Buaas,* Nichelle Gray,* Lindsey C. Krening,* Anuj Srivastava,* and Robert E. Braun*,†,1 *The Jackson Laboratory, Bar Harbor, Maine 04609 and †Graduate School of Biomedical Sciences and Engineering, The University of Maine, Orono, Maine 04469 ABSTRACT Several in vitro studies have suggested that canonical microRNA (miRNA) biogenesis requires the DICER cofactors TARBP2 and PRKRA for processing of pre-miRNAs to mature miRNAs. To investigate the roles of TARBP2 and PRKRA in miRNA biogenesis in vivo, and to determine possible functional redundancy, we first compared the phenotypes of Tarbp2 and Prkra single and double mutants. In contrast to Dicer 2/2 embryos, which die by embryonic day 7.5 (E7.5), single Tarbp2 2/2 and Prkra 2/2 mice survive beyond E7.5 and either die perinatally or survive and exhibit cranial/facial abnormalities, respectively. In contrast, only a few Tarbp2 2/2; Prkra 2/2 double mutants survived beyond E12.5, suggesting genetic redundancy between Tarbp2 and Prkra during embryonic development. Sequencing of miRNAs from single-mutant embryos at E15.5 revealed changes in abundance and isomiR type in Tarbp2 2/2, but not Prkra 2/2,embryos, demonstrating that TARBP2, but not PRKRA, functions in miRNA biogenesis of a subclass of miRNAs, and suggesting that functional redundancy between TARBP2 and PRKRA does not involve miRNA biogenesis. KEYWORDS TARBP2; PRKRA; PACT; DICER; miRNA ICRORNAS (miRNAs) are a large family of endoge- human genome is estimated to encode 1900 miRNA genes, Mnously encoded 22 nucleotide (nt) noncoding RNAs with many exhibiting developmental and stage-specificexpres- that modulate post-transcriptional gene regulation through sion patterns (Landgraf et al. 2007; Griffiths-Jones et al. 2008). translational control and the decay of messenger RNAs (mRNAs) Mammalian DICER is the principal cytoplasmic enzyme that (Krol et al. 2010). Target specificity of a miRNA is conferred by generates miRNAs and, in association with its double-stranded the seed sequence, which comprises nucleotides 2–8ofthe RNA-Binding Protein (dsRBP) cofactors TARBP2 and PRKRA, 22-nt small RNA (Lewis et al. 2003, 2005; Doench and Sharp binds the 70-nt-long pre-miRNA “hairpin” and cleaves the loop 2004; Grimson et al. 2007; Wee et al. 2012). Extensive base to produce a double-stranded RNA (dsRNA) duplex (Bernstein pairing of the miRNA seed sequence to the target mRNA leads et al. 2001; Grishok et al. 2001; Hutvagner et al. 2001; Ketting to translational repression and mRNA degradation, with 50% et al. 2001; Knight and Bass 2001; Chendrimada et al. 2005; of all protein-coding genes predicted to be miRNA targets (Baek Haase et al. 2005; Lee et al. 2006; Kok et al. 2007; Koscianska et al. 2008; Selbach et al. 2008; Friedman et al. 2009). The et al. 2011). The fully processed duplex RNA is loaded into the RNA-induced silencing complex (RISC), where one strand is Copyright © 2018 Pullagura et al. degraded leaving the guide strand (Kawamata and Tomari doi: https://doi.org/10.1534/genetics.118.300791 Manuscript received December 12, 2017; accepted for publication February 10, 2018; 2010). In TARBP2, the double-stranded RNA-binding domain published Early Online February 21, 2018. (dsRBD) motif is present in two copies along with a C-terminal Available freely online through the author-supported open access option. – “ ” This is an open-access article distributed under the terms of the Creative Commons protein protein Medipal interaction domain that binds Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), DICER (Lee et al. 2004; Laraki et al. 2008; Daniels et al. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2009). Several in vitro studies have shown that the DICER- Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10. TARBP2 complex exhibits ATP-independent dynamic diffusion 1534/genetics.118.300791/-/DC1. 1Corresponding author: The Jackson Laboratory, 600 Main St., Bar Harbor, ME 4609. along the length of pre-miRNA until it encounters a hairpin E-mail: [email protected] structure, which determines the site of cleavage by DICER Genetics, Vol. 208, 1513–1522 April 2018 1513 (Koh et al. 2012, 2017; Wilson et al. 2015). Prkra is the only was carried out as recommend by the Jackson Laboratory mammalian Tarbp2 paralog and encodes a protein with high mutant repository. sequence similarity and structure to TARBP2. Like TARBP2, PRKRA contains two dsRBDs and a C-terminal Medipal do- High-throughput sequencing of fetal small RNAs main that interacts with DICER (Laraki et al. 2008; Daniels E15.5 C57BL/6J-Tarbp2 tm1reb and C57BL/6J-Prkra lear1J em- and Gatignol 2012). In vitro biochemical and cell culture bryos were generated through timed mating intercrosses of experiments suggest that loss of the DICER cofactors TARBP2 heterozygous male and female mutant mice. Embryos were and PRKRA disrupts pre-miRNA processing, RISC loading of dissected at 15.5 days postcoitum and the associated yolk miRNAs, and RNA interference (RNAi) activity (Chendrimada sac was used for genotyping. The developmental stage was et al. 2005; Haase et al. 2005; Chakravarthy et al. 2010; Daniels confirmed using Theiler staging criteria for mouse embryo and Gatignol 2012). Apart from miRNA biogenesis, TARBP2 is development. Embryos were decapitated and both body reported to bind dsRNA directly and destabilize it (Goodarzi and head were immediately immersed in RNAlater (Thermo et al. 2014), whereas PRKRA regulates translation by activat- Fisher). Whole fetuses (head and body) were homogenized ing protein kinase (PKR) (Daniels and Gatignol 2012), a global in TRIzol followed by purification of total RNA using a inhibitor of translation. TARBP2 also promotes translation by QIAGEN miRNeasy kit (Valencia, CA). High-throughput se- binding and inhibiting PKR during human immunodeficiency quencing libraries containing small noncoding RNAs (e.g., virus infection, thereby assisting in viral replication (Daniels miRNAs or Piwi-interacting RNAs) were generated using and Gatignol 2012). 1 mg of total RNA and an Illumina TruSeq small RNA library Maternally supplied Dicer mRNA is expressed in murine preparation kit (Illumina, Inc.). Individually barcoded fetal oocytes, reaching its highest levels in germinal vesicle-stage libraries were pooled and sequenced (100 bp-end reads) on oocytes, and then falling to its lowest detectable level in a single HiSequation 2000 flow cell lane running version eight-cell-stage embryos (Murchison et al. 2007). Condi- 3 chemistry. Illumina CASAVA software was used to carry tional ablation of Dicer in oocytes using Zp3Cre results arrest out BCL to Fastq conversion. All the samples were passed in meiosis I as a consequence of spindle dysfunction and defects through quality control and sequences with poor base quali- in chromosome congression (Murchison et al. 2007; Tang et al. ties were removed. The adaptor sequences were removed 2007). Loss of zygotic Dicer mRNA, which normally begins to be using the clip_adaptors.pl module of mirdeep (v2.0.0.5) expressed in blastocyst-stage embryos (Murchison et al. 2007), (Friedlander et al. 2012). Raw reads were collapsed based leads to early developmental defects and embryonic arrest on sequence identity using the collapse_reads_md.pl module shortly after gastrulation around embryonic day 7.5 (E7.5) of mirdeep2. Collapsed reads were further annotated for (Bernstein et al. 2003). Furthermore, conditional gene targeting miRNA by mapping to miRBase version 21 (Griffiths-Jones has revealed that DICER functions in the development or homeo- et al. 2008; Kozomara and Griffiths-Jones 2011; Friedlander stasis of embryonic and fetal organs including the cardiovascular, et al. 2012) using the miraligner module of seqbuster genitourinary, musculoskeletal, and nervous systems (Bernstein (Pantano et al. 2010). IsomiRs were identified using the et al. 2003; O’Rourke et al. 2007; Saal and Harvey 2009; Zehir R isomiRs (v1.3.0) package of seqbuster. Pairwise differential et al. 2010; Small and Olson 2011). These data are consistent expression was performed among different groups using with a model where miRNAs function in the proper development DESEQ2 (v1.12.4) (Love et al. 2014). of numerous mammalian organs, and disruptions in this small RNA biogenesis pathway can result in congenital birth defects Quantitative RT-PCR and Taqman assays and in extreme cases fetal death. The mouse Tarbp2 gene en- codes a 365-amino acid protein that is localized predominantly Adult tissues were used for total RNA extraction (TRIzol; to the cytoplasm, and Tarbp2 2/2 mice on a hybrid background Invitrogen, Carlsbad, CA), followed by the production of are viable but have reduced body size and are male sterile random primed cDNA (18080051; Invitrogen). Real-time (Zhong et al. 1999). Previously characterized Prkra mutant mice PCR using SYBR green (ABI 7500; Thermo Fisher) and the are homozygous viable with cranial/facial defects (Rowe et al. 2006; ddCT method for calculating relative gene expression were Dickerman et al. 2011) and, like Tarbp2 mutants, exhibit postnatal used to determine Prkra and Tarbp2 transcript levels. b-actin growth retardation on several genetic backgrounds, including amplification was used as an endogenous control. For miRNA C57BL/6J. The similar but different phenotypes of Tarbp2 2/2 Taqman assays, total RNA is extracted (TRIzol extraction; and Prkra 2/2 mutants led us to investigate their role as DICER Invitrogen) from transformed mouse embryonic fibroblasts cofactors in vivo. (MEFs), followed by production of cDNA specific to each miRNA (4427975; Thermo Fisher) using a Taqman miRNA reverse transcription kit (4366596; Thermo Fischer). Taq- Materials and Methods man PCR using Taqman Universal Master Mix II (4440042; Thermo Fisher) and the ddCT method for calculating rela- Mouse mutants tive transcript expression were used to determine mature Tarbp2tm1reb (Zhong et al.
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