MECP2 Is Post-transcriptionally Regulated during Human Neurodevelopment by Combinatorial Action of RNA-Binding Proteins and miRNAs

Rodrigues, Deivid C.; Kim, Dae Sung; Yang, Guang; Zaslavsky, Kirill; Ha, Kevin C.H.; Mok, Rebecca S.F.; Ross, P. Joel; Zhao, Melody; Piekna, Alina; Wei, Wei; Blencowe, Benjamin J.; Morris, Quaid; Ellis, James

doi:10.1016/j.celrep.2016.09.049

Cited by 63 publications

(76 citation statements)

References 33 publications

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“…Enrichment was evident, though diminished, for sites with weaker predicted affinities, for ∆∆G values up to ~3 kcal/mol. This enrichment is consistent with the established functional roles for PUM2 via 3´ UTR binding ((Bohn et al, 2018;Kedde et al, 2010;Miles et al, 2012;Rodrigues et al, 2016;Wickens et al, 2002)).…”

Section: Assessing the Thermodynamic Model For Pum2 Rna Occupancies Isupporting

confidence: 88%

“…PUF family proteins ( Figure 1A) are universal in eukaryotes and have been implicated in regulation of mRNA turnover, transport, translation, and localization (Miller and Olivas, 2011;Quenault et al, 2011). In mammals, PUF proteins play important roles in brain and germline development, regulation of innate immunity and other processes (Bohn et al, 2018;Chen et al, 2012;Gennarino et al, 2018;Gennarino et al, 2015;Kedde et al, 2010;Liu et al, 2017;Miles et al, 2012;Rodrigues et al, 2016;Tichon et al, 2016;Van Etten et al, 2012;Vessey et al, 2010). Extensive prior biochemical, structural, evolutionary, and in-vivo studies of PUF proteins provide a powerful starting point for our quantitative and systematic dissection of specificity (Figure S1A & references therein) and allow us to pose specific biological, engineering, and biophysical questions.…”

Section: Introductionmentioning

confidence: 99%

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A quantitative and predictive model for RNA binding by human Pumilio proteins

Jarmoskaite

Denny

Vaidyanathan

et al. 2018

Preprint

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High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA/RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies. v8.0 3 Results Library designStarting with the PUM2 consensus motif, which has been determined by pull-down, crosslinking and in vitro selection experiments ( Figures 1A, S1A), we designed an oligonucleotide v8.0 5 library to systematically address the factors that determine binding specificity ( Figure S1B). To control for structural and context effects, each sequence variant was embedded in two to four scaffolds ( Figure 1B). We systematically varied the sequence of the PUM2 binding site and the flanking sequence ( Figure S1B). We also included insertions to test the potential for noncontiguous binding sites and variants of sequence motifs of related PUF proteins to provide additional sequence variation for testing PUM2 binding models ( Figure S1B). Massively parallel measurements of PUM2 binding affinitiesUsing RNA-MaP, we determined PUM2 protein binding affinities for >20,000 distinct RNAs and we report on >5000 herein; sequences designed to address distinct questions will be reported separately. The DNA library was sequenced on an Illumina MiSeq flow cell, followed by in situ transcription in a custom-built imaging and fluidics setup ( Figure 1C; ). The RNA transcripts were immobilized by stalling the RNA polymerase at the end of the DNA template, and RNA-protein association was measured by equilibrating the RNA with increasing concentrations of fluorescently labeled protein and imaging binding to each cluster (comprising ~1000 copies of an RNA variant) (Buenrostro et al., 2014)( Figure 1C). The resulting binding curves were used to obtain the dissociation constant (KD) and the corresponding ∆G value (= RTlnKD) of the protein for each RNA variant.

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Section: Assessing the Thermodynamic Model For Pum2 Rna Occupancies Isupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

A quantitative and predictive model for RNA binding by human Pumilio proteins

Jarmoskaite

Denny

Vaidyanathan

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…This exon inclusion therefore introduces a uORF into the mRNA, which downregulates protein production ( Figure 4B, (Kriaucionis and Bird, 2004). The 3′ UTR of MECP2 is also alternatively processed, leading to various short (~100 nt) or long (~8 kb) forms, which are differentially translated and differentially expressed during neuronal differentiation (Rodrigues et al, 2016). We observed the impact of both of these transcript processing events in 50-day old human neural cultures.…”

Section: Transcript-specific Translation During Human Neuronal Differmentioning

confidence: 84%

Widespread translational remodeling during human neuronal differentiation

Blair

Hockemeyer

Doudna

et al. 2017

Preprint

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Faithful cellular differentiation requires precise coordination of changes in gene expression. However, the relative contributions of transcriptional and translational regulation during human cellular differentiation are unclear. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and characterized genomewide RNA and translation levels during neurogenesis. We find that thousands of genes change at the translation level across differentiation without a corresponding change in RNA level. Specifically, we identify mTOR complex 1 signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by transcript 3′ UTRs, through regulatory sequences. Together, our findings identify a functional role for the dramatic 3′ UTR extensions that occur during brain development, and provide insights to interpret genetic variants in post-transcriptional control factors that influence neurodevelopmental disorders and diseases.

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“…This exon inclusion therefore introduces a uORF into the mRNA, which downregulates protein production (Figure 4B, (Kriaucionis and Bird, 2004). The 3′ UTR of MECP2 is also alternatively processed, leading to various short (~100 nt) or long (~8 kb) forms, which are differentially translated and differentially expressed during neuronal differentiation (Rodrigues et al, 2016). We observed the impact of both of these transcript processing events in 50-day old human neuronal cultures.…”

Section: Resultsmentioning

confidence: 99%

Widespread Translational Remodeling during Human Neuronal Differentiation

et al. 2017

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Summary Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We find that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identify mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3′ UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation, and a crucial role of 3′ UTRs in driving cell-type specific translation.

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MECP2 Is Post-transcriptionally Regulated during Human Neurodevelopment by Combinatorial Action of RNA-Binding Proteins and miRNAs

Cited by 63 publications

References 33 publications

A quantitative and predictive model for RNA binding by human Pumilio proteins

A quantitative and predictive model for RNA binding by human Pumilio proteins

Widespread translational remodeling during human neuronal differentiation

Widespread Translational Remodeling during Human Neuronal Differentiation

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