Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing

Gangras, Pooja; Dayeh, Daniel M.; Mabin, Justin W.; Nakanishi, K.; Singh, Guramrit

doi:10.1007/978-1-4939-7339-2_1

Cited by 9 publications

(9 citation statements)

References 20 publications

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“…For RNA-seq libraries, 5 μg of total cellular RNA was depleted of ribosomal RNA (RiboZero kit, Illumina), and subjected to base hydrolysis. RNA fragments were then used to generate strand-specific libraries using a custom library preparation method (Gangras et al, 2018). Briefly, a pre-adenylated miR-Cat33 DNA adaptor was ligated to RNA 3′ ends and used as a primer binding site for reverse-transcription (RT) using a special RT primer.…”

Section: Methodsmentioning

confidence: 99%

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

et al. 2018

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SUMMARY The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that the EJC originates as an SR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. The switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

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Section: Methodsmentioning

confidence: 99%

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

et al. 2018

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“…For RNA-Seq libraries, 5 μg of total cellular RNA was depleted of ribosomal RNA (RiboZero kit, Illumina), and subjected to base hydrolysis. RNA fragments were then used to generate strand-specific libraries using a custom library preparation method [93]. Briefly, a pre-adenylated miR-Cat33 DNA adapter was ligated to RNA 3 0 -ends and used as a primer binding site for reverse-transcription (RT) using a special RT primer.…”

Section: Rip-seq and Rna-seq Library Preparationmentioning

confidence: 99%

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3′UTR intron-containing NMD targets

et al. 2020

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Many post-transcriptional mechanisms operate via mRNA 3 0 UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination � 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3 0 UTR introns (3 0 UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3 0 UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3 0 UI in zebrafish, mouse and humans. foxo3b is one such proximal 3 0 UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3 0 UIs may serve gene regulatory function in vertebrates.

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“…For RNA-Seq libraries, 5 µg of total cellular RNA was depleted of ribosomal RNA (RiboZero kit, Illumina), and subjected to base hydrolysis. RNA fragments were then used to generate strand-specific libraries using a custom library preparation method (77). Briefly, a pre-adenylated miR-Cat33 DNA adapter was ligated to RNA 3’-ends and used as a primer binding site for reverse-transcription (RT) using a special RT primer.…”

Section: Methodsmentioning

confidence: 99%

Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

Gangras

Patton

et al. 2019

Preprint

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17The Exon Junction Complex (EJC) regulates many steps in post-transcriptional gene 18 expression and is essential for cellular function and organismal development; however, 19 EJC-regulated genes and genetic pathways during development remain largely 20 unknown. To study EJC function during zebrafish development, we first established that 21 zebrafish EJCs mainly bind ~24 nucleotides upstream of exon-exon junctions, and are 22 also detected at more distant non-canonical positions. We then generated mutations in 23 two zebrafish EJC core genes, rbm8a and magoh, and observed that homozygous 24 mutant embryos show paralysis, muscle disorganization, neural cell death, and motor 25 neuron outgrowth defects. Coinciding with developmental defects, mRNAs subjected to 26 Nonsense-Mediated mRNA Decay (NMD) due to translation termination ≥ 50 nts 27 upstream of the last exon-exon junction are upregulated in EJC mutant embryos.28 Surprisingly, several transcripts containing 3′UTR introns (3′UI) < 50 nts downstream of 29 a stop codon are also upregulated in EJC mutant embryos. These proximal containing transcripts are also upregulated in NMD-compromised zebrafish embryos, 31 cultured human cells, and mouse embryonic stem cells. Loss of function of foxo3b, one 32 of the upregulated proximal 3′UI-containing genes, partially rescues EJC mutant motor 33 neuron outgrowth. In addition to foxo3b, 166 other genes contain a proximal 3′UI in 34 zebrafish, mouse and humans, and these genes are enriched in nervous system 35 development and RNA binding functions. A proximal 3′UI-containing 3′UTR from one of 36 these genes, HNRNPD, is sufficient to reduce steady state transcript levels when fused 37 to a β-globin reporter in HeLa cells. Overall, our work shows that genes with stop 38 codon-proximal 3′UIs encode a new class of EJC-regulated NMD targets with critical 39 roles during vertebrate development.40 84 suggesting that 3′UIs may play an important role in regulating tissue-specific 85 developmental programs via EJC-dependent NMD. However, specific mRNAs, genetic 86 5 pathways, and developmental events that are regulated by 3′UI-dependent NMD 87 remain largely unknown. 88In this work, we establish zebrafish as a model to study EJC developmental 89 functions. As expected from work in human cells, we show that in zebrafish embryos 90 the EJC mainly binds ~24 nts upstream of exon-exon junctions and is also detected on 91 more distant exonic positions. Zebrafish Rbm8a and Magoh are crucial for early 92 zebrafish embryonic development, with muscle and neural lineages being particularly 93 sensitive to the loss of EJC. We find that EJC-dependent NMD is disrupted in rbm8a 94 and magoh mutant embryos. Strikingly, we uncover a class of genes that contain a stop 95 codon-proximal 3′UI (intron within 50 nts of the stop codon) and are upregulated in 96 zebrafish EJC mutant embryos and upf1 morphants. We show that loss of foxo3b, a 97 proximal 3′UI-containing gene whose transcript and protein levels are elevated in EJC 98 mutants, partially...

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Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing

Cited by 9 publications

References 20 publications

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3′UTR intron-containing NMD targets

Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

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