Genetic Characterization of smg-8 Mutants Reveals No Role in C. elegans Nonsense Mediated Decay

The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3′ untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.Molecular and Cellular Biolog

Developmental Bias in Cleavage-Stage Mouse Blastomeres

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.Molecular and Cellular Biolog

Developmental Bias in Cleavage-Stage Mouse Blastomeres

The cleavage stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, as preimplantation genetic diagnosis (PGD) requires removal of blastomeres from the early human embryo. To determine if blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, non-invasive combinatorial fluorescent labeling method for embryonic lineage tracing

Author Correction:Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts (Nature Genetics, (2021), 53, 1, (86-99), 10.1038/s41588-020-00750-6)

This paper was originally published without open access. As of the date of this correction, the paper is available online as an open-access paper under a Creative Commons Attribution 4.0 International License. *Lists of authors and their affiliations appear online

Modulation of pha-4/FoxA and C. elegans Foregut Development by the Novel Gene smg-8

FoxA transcription factors are central regulators of gut development in all species. In C. elegans, pha-4/FoxA is necessary to generate cells of the foregut, or pharynx. FoxA factors need to be precisely regulated for proper development, yet we know very little about FoxA regulation. To look for potential genes that act as pha-4 regulators, the Mango lab previously conducted two screens for suppressors of the lethality associated with a partial loss of pha-4 activity. Both screens uncovered smg-8, a novel gene that is highly conserved amongst metazoans. Interestingly, the human homolog of smg-8 is amplified in some breast cancers, which also depend on FoxA1. This observation makes smg-8 a very exciting gene to investigate. The goal of my thesis is to analyze smg-8 to better understand its function and potential role as a candidate regulator of pha-4/FoxA, using C. elegans as a model system. In this thesis, I show that C. elegans smg-8 does not have a role in the Nonsense Mediated Decay pathway. I find that smg-8 modulates pha-4 protein levels during embryonic development. This work is the first direct evidence that smg-8 is a modulator of pha-4. I used biochemical and bioinformatic approaches to uncover possible partners of smg-8. These approaches identified several interesting candidates that will help place C. elegans smg-8 in a functional pathway. This work has expanded our understanding of smg-8 function and lays the foundation for further investigation of the role of this novel gene as a regulator of pha-4/FoxA in C. elegans

<i>smg-8</i> lacks an NMD phenotype for the native NMD target <i>rpl-12</i>.

<p>(A) RT-PCR was performed using a pair of primers that distinguish the two spliced isoforms of <i>rpl-12</i>; the upper, PTC band is visible only when the NMD pathway is compromised by <i>smg-1, smg-2</i> or sm<i>g-3</i> mutations (lanes 2, 3 and 4). Only the lower, WT band is observed in wild-type (lane 1) and <i>smg-8</i> mutant (lane 5) animals. (B) Wild-type worms were fed bacteria expressing dsRNA targeting <i>smg-1</i>, <i>smg-8</i> or <i>smg-9</i> from the Ahringer dsRNA library <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Kamath1" target="_blank">[26]</a>. RNA was analyzed as in (A). (C) As in (B), using an enhanced RNAi mutant <i>eri-6/7 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Zhuang1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Fischer1" target="_blank">[23]</a>. (D) As in (B), using the <i>smg-8(tm2937)</i> mutant strain.</p

<i>smg-8</i> lacks the vulva phenotype associated with mutations in other NMD genes.

<p>(A) Schematic representation of the <i>tm2937</i> allele, which contains a 272 bp deletion and a 1 bp insertion. This deletion encompasses 22 bp upstream of the start site and the first two exons. Arrows indicate primers used for RT-qPCR (B) Vulval protrusion is one of the phenotypes of canonical <i>smg</i> genes. Left panel shows a wildtype vulva, middle panel shows a <i>smg-8(tm2937)</i> mutant and right panel shows a <i>smg-1(r861)</i> mutant. <i>smg-8</i> mutants are similar to wild-type and not to <i>smg-1</i>. Arrowheads denote the vulva.</p

<i>smg-8</i> does not restore expression of <i>myo-3:</i>:GFP, an exogenous NMD target.

<p>(A) Schematic representation of the exogenous NMD GFP reporter, driven by the <i>myo-3</i> promoter, which is destabilized by an amino acid sequence that marks a protein for degradation (degron), and a long 3′UTR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Link1" target="_blank">[24]</a>. (B) <i>smg-8</i> and control mutations were introduced into CL724 (<i>myo-3</i>::GFP) worms. The double combinations were then inspected under a fluorescent microscope. <i>smg-1</i> and <i>smg-3</i> mutants express high levels of GFP. In contrast, <i>smg-8</i> animals photographed under the same conditions show only a slight accumulation of GFP, similar to the wild type.</p

<i>smg-8</i> lacks an NMD phenotype for the native NMD target <i>rpl-7a</i>.

<p>(A) Schematic representation of the two alternatively spliced isoforms of <i>rpl-7a.</i> The isoform containing the premature termination codon (PTC) is subject to degradation by NMD, whereas the shorter isoform is not. RT-PCR was performed using a pair of primers that distinguish the two spliced isoforms (purple arrows). (B) The upper, PTC band is visible only when the NMD pathway is compromised by <i>smg-1, smg-2</i> or sm<i>g-3</i> mutations (lanes 2, 3 and 4). Only the lower WT band is observed in wild-type (lane 1) and <i>smg-8</i> mutant (lane 5) animals. (C) Wild-type worms were fed bacteria expressing dsRNA targeting <i>smg-1</i>, <i>smg-8</i> or <i>smg-9</i> from the Ahringer dsRNA library <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Kamath1" target="_blank">[26]</a>. RNA was analyzed as in (B). (D) An enhanced RNAi mutant strain <i>eri-6/7 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Zhuang1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Fischer1" target="_blank">[23]</a> was used and RNAi conducted as in (C). RNA was analyzed as in (B). (E) As in D, using the <i>smg-8(tm2937)</i> mutant strain. (F) RT-qPCR using primers flanking the PTC-containing isoform of <i>rpl-7a,</i> mRNA levels were calculated using the delta-delta-CT method, relative to the control gene <i>pmp-3 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Hoogewijs1" target="_blank">[27]</a>. Fold enrichment of the PTC mRNA was normalized to 1 for wild-type. The <i>smg-1</i> and <i>smg-3</i> mutants show an enrichment of 15 and 38 fold, respectively. In contrast, in <i>smg-8</i> mutants, the accumulation of the PTC containing isoform is similar to wild-type (0.7 fold enrichment). <i>smg-8</i> and <i>eri-6/7</i> mutant worms treated with <i>smg-8</i> RNAi show 0.7 and 0.4 fold enrichment, respectively. (G) RT-qPCR to quantify <i>smg-8</i> RNA. mRNA levels were calculated using the delta-delta-CT method, relative to the control gene <i>pmp-3 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049490#pone.0049490-Hoogewijs1" target="_blank">[27]</a>. Fold enrichment was normalized to 1 for wild-type. <i>smg-8</i> and <i>eri-6/7</i> worms treated with <i>smg-8</i> RNAi show 0.3 and 0.26 fold enrichment, respectively. (H) As in (G) for wild-type animals and a negative control that lacked Reverse Transcriptase (No RT). Fold enrichment was normalized to 1 for wild-type. No RT control shows 0.3 fold enrichment.</p