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Dynamic regulation of mRNA decay during neural development.

Burow DA, Umeh-Garcia MC, True MB, Bakhaj CD, Ardell DH, Cleary MD - Neural Dev (2015)

Bottom Line: A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis.We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.

View Article: PubMed Central - PubMed

Affiliation: Quantitative and Systems Biology Graduate Program, University of California, 5200 N. Lake Rd, Merced, CA, USA. dburow@ucmerced.edu.

ABSTRACT

Background: Gene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay.

Results: We developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.

Conclusions: We found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.

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Pros > UPRT TU-tagging purifies neural mRNAs from embryos. (A)Prospero-GAL4 driving UAS-GFP::lacZ.nls (encoding a nuclear-localized GFP-β-galactosidase fusion) in the embryonic central nervous system shows the expression pattern of prospero-GAL4. Anti-Prospero = magenta, anti-β-galactosidase = green. (B) RNA in situ hybridization for eight genes identified as nervous system-enriched by TU-tagging. Gene annotation symbols are listed in the top panel. Top panel = ventral view. Bottom panel = lateral view. Developmental stage is listed in the lower right corner of each embryo image. (C) Relative abundance of mesoderm or muscle-specific mRNAs and neural-specific mRNAs in 1-hour pulse-labeled TU-RNA samples. ** = FDR ≤0.20, * = FDR ≤0.40 based on SAM analysis. (D) Gene ontology categories depleted from neural TU-RNA samples (mRNAs below detection in the 1-hour pulse pros > UPRT TU-RNA microarrays but present in 1-hour pulse whole embryo TU-RNA microarrays). All categories were significantly depleted at P values ≤ 1.0 × 10−4 based on DAVID analysis.
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Fig2: Pros > UPRT TU-tagging purifies neural mRNAs from embryos. (A)Prospero-GAL4 driving UAS-GFP::lacZ.nls (encoding a nuclear-localized GFP-β-galactosidase fusion) in the embryonic central nervous system shows the expression pattern of prospero-GAL4. Anti-Prospero = magenta, anti-β-galactosidase = green. (B) RNA in situ hybridization for eight genes identified as nervous system-enriched by TU-tagging. Gene annotation symbols are listed in the top panel. Top panel = ventral view. Bottom panel = lateral view. Developmental stage is listed in the lower right corner of each embryo image. (C) Relative abundance of mesoderm or muscle-specific mRNAs and neural-specific mRNAs in 1-hour pulse-labeled TU-RNA samples. ** = FDR ≤0.20, * = FDR ≤0.40 based on SAM analysis. (D) Gene ontology categories depleted from neural TU-RNA samples (mRNAs below detection in the 1-hour pulse pros > UPRT TU-RNA microarrays but present in 1-hour pulse whole embryo TU-RNA microarrays). All categories were significantly depleted at P values ≤ 1.0 × 10−4 based on DAVID analysis.

Mentions: We previously used TU-tagging to identify taranis as a nervous system-expressed gene in Drosophila embryos [19]. These experiments used prospero-GAL4 to drive UAS-T.g.UPRT expression (pros > UPRT). Embryos at 0 to 16 h of development were exposed to 4-thiouracil (4TU) for 2 h and TU-tagged RNA was isolated from whole embryo lysates. Purified TU-RNA was compared to non-tagged RNA from the same embryos in microarray experiments. The prospero-GAL4 construct expresses GAL4 in neuroblasts, ganglion mother cells, and glia of the central nervous system (CNS) (Figure 2A) in addition to peripheral sensory neurons. Prospero-Gal4 driving UAS-T.g.UPRT also results in mRNA tagging in post-mitotic CNS neurons since the UPRT (and likely some converted 4-thio-UMP) is transferred from progenitors to neurons [20].Figure 2


Dynamic regulation of mRNA decay during neural development.

Burow DA, Umeh-Garcia MC, True MB, Bakhaj CD, Ardell DH, Cleary MD - Neural Dev (2015)

Pros > UPRT TU-tagging purifies neural mRNAs from embryos. (A)Prospero-GAL4 driving UAS-GFP::lacZ.nls (encoding a nuclear-localized GFP-β-galactosidase fusion) in the embryonic central nervous system shows the expression pattern of prospero-GAL4. Anti-Prospero = magenta, anti-β-galactosidase = green. (B) RNA in situ hybridization for eight genes identified as nervous system-enriched by TU-tagging. Gene annotation symbols are listed in the top panel. Top panel = ventral view. Bottom panel = lateral view. Developmental stage is listed in the lower right corner of each embryo image. (C) Relative abundance of mesoderm or muscle-specific mRNAs and neural-specific mRNAs in 1-hour pulse-labeled TU-RNA samples. ** = FDR ≤0.20, * = FDR ≤0.40 based on SAM analysis. (D) Gene ontology categories depleted from neural TU-RNA samples (mRNAs below detection in the 1-hour pulse pros > UPRT TU-RNA microarrays but present in 1-hour pulse whole embryo TU-RNA microarrays). All categories were significantly depleted at P values ≤ 1.0 × 10−4 based on DAVID analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Pros > UPRT TU-tagging purifies neural mRNAs from embryos. (A)Prospero-GAL4 driving UAS-GFP::lacZ.nls (encoding a nuclear-localized GFP-β-galactosidase fusion) in the embryonic central nervous system shows the expression pattern of prospero-GAL4. Anti-Prospero = magenta, anti-β-galactosidase = green. (B) RNA in situ hybridization for eight genes identified as nervous system-enriched by TU-tagging. Gene annotation symbols are listed in the top panel. Top panel = ventral view. Bottom panel = lateral view. Developmental stage is listed in the lower right corner of each embryo image. (C) Relative abundance of mesoderm or muscle-specific mRNAs and neural-specific mRNAs in 1-hour pulse-labeled TU-RNA samples. ** = FDR ≤0.20, * = FDR ≤0.40 based on SAM analysis. (D) Gene ontology categories depleted from neural TU-RNA samples (mRNAs below detection in the 1-hour pulse pros > UPRT TU-RNA microarrays but present in 1-hour pulse whole embryo TU-RNA microarrays). All categories were significantly depleted at P values ≤ 1.0 × 10−4 based on DAVID analysis.
Mentions: We previously used TU-tagging to identify taranis as a nervous system-expressed gene in Drosophila embryos [19]. These experiments used prospero-GAL4 to drive UAS-T.g.UPRT expression (pros > UPRT). Embryos at 0 to 16 h of development were exposed to 4-thiouracil (4TU) for 2 h and TU-tagged RNA was isolated from whole embryo lysates. Purified TU-RNA was compared to non-tagged RNA from the same embryos in microarray experiments. The prospero-GAL4 construct expresses GAL4 in neuroblasts, ganglion mother cells, and glia of the central nervous system (CNS) (Figure 2A) in addition to peripheral sensory neurons. Prospero-Gal4 driving UAS-T.g.UPRT also results in mRNA tagging in post-mitotic CNS neurons since the UPRT (and likely some converted 4-thio-UMP) is transferred from progenitors to neurons [20].Figure 2

Bottom Line: A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis.We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.

View Article: PubMed Central - PubMed

Affiliation: Quantitative and Systems Biology Graduate Program, University of California, 5200 N. Lake Rd, Merced, CA, USA. dburow@ucmerced.edu.

ABSTRACT

Background: Gene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. Such information should reveal the degree to which mRNA decay contributes to differential gene expression and provide a foundation for identifying regulatory mechanisms that affect neural mRNA decay.

Results: We developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. Our approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. We also identified correlations between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. We found that decreased expression of the RNA-binding protein Pumilio stabilized predicted neural mRNA targets and that a PRE is necessary to trigger reporter-transcript decay in the nervous system.

Conclusions: We found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. We found that Pumilio is one component of this network, revealing a novel function for this RNA-binding protein.

Show MeSH
Related in: MedlinePlus