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1.
Mol Cell ; 83(24): 4445-4460.e7, 2023 Dec 21.
Article in English | MEDLINE | ID: mdl-37995689

ABSTRACT

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.


Subject(s)
Drosophila Proteins , Transcription Termination, Genetic , Animals , RNA , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Small Nuclear/genetics , Transcription Factors/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster
2.
Methods ; 196: 121-128, 2021 12.
Article in English | MEDLINE | ID: mdl-33882363

ABSTRACT

Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.


Subject(s)
Polyadenylation , RNA, Circular , Biomarkers , Polyadenylation/genetics , RNA/genetics , RNA/metabolism , RNA Splicing/genetics , RNA, Circular/genetics
3.
Methods Mol Biol ; 2209: 321-332, 2021.
Article in English | MEDLINE | ID: mdl-33201478

ABSTRACT

Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs that have covalently linked ends. These transcripts are resistant to degradation by exonucleases, which enables some to accumulate to higher levels than the associated linear mRNA. In general, exonic circular RNAs accumulate in the cytoplasm, but functions for most of these transcripts remain unknown. It has been proposed that some may modulate the activity of microRNAs or RNA-binding proteins, be translated to yield protein products, or regulate innate immune responses. Recent work has revealed that circular RNAs are exported from the nucleus in a length-dependent manner and that the subcellular localization of these transcripts can be controlled by the DExH/D-box helicase Hel25E in Drosophila. Here, we describe how RNAi screening combined with subcellular fractionation and quantitative reverse transcription PCR (RT-qPCR) can be used to identify regulators of circular RNA localization in Drosophila cells. Long double-stranded RNAs (dsRNAs) that activate the RNA interference (RNAi) pathway are used to deplete factors of interest followed by biochemical fractionation to separate nuclear and cytoplasmic RNAs. RT-qPCR primers that amplify across the backsplicing junction of specific circular RNAs are then used to quantify the relative amounts of these transcripts in the nuclear and cytoplasmic compartments. In total, this approach can be broadly used to characterize circular RNA nuclear export and localization mechanisms, including to identify novel regulatory factors and their breadth of circular RNA targets.


Subject(s)
DEAD-box RNA Helicases/metabolism , Drosophila Proteins/metabolism , RNA Transport , RNA, Circular/metabolism , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Cell Nucleus/metabolism , Cytoplasm/metabolism , Drosophila melanogaster/metabolism , RNA Interference , RNA Precursors/metabolism
4.
Trends Biochem Sci ; 45(11): 923-934, 2020 11.
Article in English | MEDLINE | ID: mdl-32800671

ABSTRACT

The Integrator complex is conserved across metazoans and controls the fate of many nascent RNAs transcribed by RNA polymerase II (RNAPII). Among the 14 subunits of Integrator is an RNA endonuclease that is crucial for the biogenesis of small nuclear RNAs and enhancer RNAs. Integrator is further employed to trigger premature transcription termination at many protein-coding genes, thereby attenuating gene expression. Integrator thus helps to shape the transcriptome and ensure that genes can be robustly induced when needed. The molecular functions of Integrator subunits beyond the RNA endonuclease remain poorly understood, but some can act independently of the multisubunit complex. We highlight recent molecular insights into Integrator and propose how misregulation of this complex may lead to developmental defects and disease.


Subject(s)
RNA Polymerase II , RNA , Animals , Humans , RNA/genetics , RNA/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Transcription, Genetic/genetics
6.
Mol Cell ; 76(5): 738-752.e7, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31809743

ABSTRACT

The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25- to 60-nt-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, 15%-20% of genes display highly unstable promoter Pol II, suggesting that paused polymerase might dissociate from template DNA at these promoters and release a short, non-productive mRNA. Here, we report that paused Pol II can be actively destabilized by the Integrator complex. Specifically, we present evidence that Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and drive termination of paused Pol II. These findings uncover a previously unappreciated mechanism of metazoan gene repression, akin to bacterial transcription attenuation, wherein promoter-proximal Pol II is prevented from entering productive elongation through factor-regulated termination.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA, Messenger/biosynthesis , Transcription Elongation, Genetic , Animals , Cell Line , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , RNA Polymerase II/genetics , RNA, Messenger/genetics
7.
Genes Dev ; 33(21-22): 1525-1538, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31530651

ABSTRACT

Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many noncatalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.


Subject(s)
Drosophila Proteins/genetics , Drosophila/genetics , Gene Expression Regulation , Metallothionein/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/metabolism , Animals , Cell Line , Copper/toxicity , Endoribonucleases/metabolism , Gene Expression Regulation/drug effects , Protein Binding , RNA Cleavage , Stress, Physiological/drug effects
8.
Article in English | MEDLINE | ID: mdl-32086332

ABSTRACT

A complex network of RNA transcripts is generated from eukaryotic genomes, many of which are processed in unexpected ways. Here, we highlight how premature transcription termination events at protein-coding gene loci can simultaneously lead to the generation of short RNAs and attenuate production of full-length mRNA transcripts. We recently showed that the Integrator (Int) complex can be selectively recruited to protein-coding gene loci, including Drosophila metallothionein A (MtnA), where the IntS11 RNA endonuclease cleaves nascent transcripts near their 5' ends. Such premature termination events catalyzed by Integrator can repress the expression of some full-length mRNAs by more than 100-fold. Transcription at small nuclear RNA (snRNA) loci is likewise terminated by Integrator cleavage, but protein-coding and snRNA gene loci have notably distinct dependencies on Integrator subunits. Additional mechanisms that attenuate eukaryotic gene outputs via premature termination have been discovered, including by the cleavage and polyadenylation machinery in a manner controlled by U1 snRNP. These mechanisms appear to function broadly across the transcriptome. This suggests that synthesis of full-length transcripts is not always the default option and that premature termination events can lead to a variety of transcripts, some of which may have important and unexpected biological functions.

9.
Genes Dev ; 32(9-10): 639-644, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29773557

ABSTRACT

Circular RNAs (circRNAs) are generated from many protein-coding genes. Most accumulate in the cytoplasm, but how circRNA localization or nuclear export is controlled remains unclear. Using RNAi screening, we found that depletion of the Drosophila DExH/D-box helicase Hel25E results in nuclear accumulation of long (>800-nucleotide), but not short, circRNAs. The human homologs of Hel25E similarly regulate circRNA localization, as depletion of UAP56 (DDX39B) or URH49 (DDX39A) causes long and short circRNAs, respectively, to become enriched in the nucleus. These data suggest that the lengths of mature circRNAs are measured to dictate the mode of nuclear export.


Subject(s)
Active Transport, Cell Nucleus/genetics , Drosophila melanogaster/genetics , RNA/genetics , RNA/metabolism , Amino Acid Sequence , Amino Acids/genetics , Animals , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Evolution, Molecular , Genetic Variation , HeLa Cells , Humans , Protein Transport/genetics , RNA, Circular
10.
Mol Cell ; 68(5): 940-954.e3, 2017 Dec 07.
Article in English | MEDLINE | ID: mdl-29174924

ABSTRACT

Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.


Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/genetics , RNA Precursors/biosynthesis , RNA Splicing , RNA, Messenger/biosynthesis , RNA/biosynthesis , Spliceosomes/genetics , Transcription, Genetic , Animals , Cell Line , Drosophila Proteins/biosynthesis , Drosophila melanogaster/metabolism , Laccase/biosynthesis , Laccase/genetics , RNA/genetics , RNA Interference , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , RNA Stability , RNA, Circular , RNA, Messenger/genetics , Ribonucleoproteins, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/metabolism , Spliceosomes/metabolism , Transcription Termination, Genetic , Transfection
11.
Methods Mol Biol ; 1648: 143-154, 2017.
Article in English | MEDLINE | ID: mdl-28766295

ABSTRACT

Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs. Because they have covalently linked ends, circular RNAs are resistant to degradation by exonucleases and some accumulate to higher levels than their associated linear mRNAs. The functions of most circular RNAs are still unknown, but recent work has revealed key insights into how the pre-mRNA splicing machinery catalyzes backsplicing. Exons that circularize are often flanked by intronic repeat sequences that are complementary to one another, and backsplicing is triggered when these repeats base pair and bring the intervening splice sites into close proximity. Here, we describe how this knowledge has been translated into a simple plasmid-based method for ectopically expressing circular RNAs in eukaryotic cells. The sequence of interest is cloned into an artificial exon that is flanked by complementary intronic repeats. The plasmid is then transfected into cells, transcription is induced, and the cellular splicing machinery generates the desired circular RNA. Total RNA is isolated and the efficiency/specificity of circular RNA biogenesis is validated by Northern blot analysis. Beyond allowing overexpression of natural circular RNAs to define their functions, this approach can be used to produce designer RNA circles that are translated or bind specific cellular factors, such as microRNAs or proteins.


Subject(s)
Gene Expression , Plasmids/genetics , RNA Splicing , RNA, Messenger/genetics , Animals , Cell Line , Drosophila melanogaster , Plasmids/metabolism , RNA, Messenger/biosynthesis
12.
Mol Cell ; 66(1): 1-2, 2017 Apr 06.
Article in English | MEDLINE | ID: mdl-28388436

ABSTRACT

In this issue of Molecular Cell, Legnini et al. (2017) and Pamudurti et al. (2017) demonstrate that endogenous circular RNAs may generate proteins, thereby expanding the eukaryotic proteome and revealing novel modes of cap-independent translation.


Subject(s)
Proteins , Ribosomes , Humans , Protein Biosynthesis , RNA, Messenger
13.
Mol Cell ; 64(2): 416-430, 2016 10 20.
Article in English | MEDLINE | ID: mdl-27768875

ABSTRACT

Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in ∼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.


Subject(s)
Chromatin/chemistry , Mouse Embryonic Stem Cells/metabolism , Nuclear Proteins/chemistry , Proteome/chemistry , RNA, Untranslated/chemistry , RNA-Binding Proteins/chemistry , Animals , Binding Sites , Chromatin/metabolism , Chromatin/radiation effects , Gene Expression , HEK293 Cells , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/radiation effects , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Peptide Mapping/methods , Photochemical Processes , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Proteome/genetics , Proteome/metabolism , Proteomics/methods , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ultraviolet Rays
14.
J Cell Biol ; 213(5): 557-70, 2016 06 06.
Article in English | MEDLINE | ID: mdl-27241916

ABSTRACT

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3' processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3' end processing with transcription termination.


Subject(s)
Drosophila melanogaster/genetics , Genetic Loci , Histones/genetics , RNA Precursors/genetics , RNA Processing, Post-Transcriptional/genetics , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Histones/metabolism , In Situ Hybridization, Fluorescence , Models, Biological , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Phenotype , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U7 Small Nuclear/metabolism , Transgenes
15.
Genes Dev ; 29(20): 2168-82, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26450910

ABSTRACT

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.


Subject(s)
Drosophila melanogaster/genetics , Gene Expression Regulation, Enzymologic , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Introns/genetics , Laccase/biosynthesis , Laccase/genetics , RNA/genetics , Animals , Base Pairing , Drosophila Proteins/genetics , Humans , Microsatellite Repeats/genetics , Nerve Tissue Proteins/genetics , Receptors, Cell Surface/genetics , Serine-Arginine Splicing Factors/genetics
16.
RNA ; 21(7): 1375-89, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26015596

ABSTRACT

Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3' ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3' ends contain post-transcriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3' end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3' ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nt at the 3' end of mature histone mRNA maintaining the length of the histone transcripts. Histone mRNAs in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3' additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Animals , Base Sequence , Cells, Cultured , DNA Primers , DNA, Complementary/genetics , Drosophila , Histones/genetics , Humans , Polyadenylation , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
17.
Dev Cell ; 32(3): 373-86, 2015 Feb 09.
Article in English | MEDLINE | ID: mdl-25669886

ABSTRACT

Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.


Subject(s)
Chromatin/genetics , Histones/metabolism , Multigene Family/genetics , Protein Processing, Post-Translational/physiology , Animals , DNA Replication/genetics , Drosophila , Epigenesis, Genetic/genetics , Histone-Lysine N-Methyltransferase/metabolism , Methylation
18.
Nucleus ; 5(6): 613-25, 2014.
Article in English | MEDLINE | ID: mdl-25493544

ABSTRACT

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.


Subject(s)
Drosophila Proteins/biosynthesis , Embryonic Development , Histones/genetics , Polyadenylation/genetics , mRNA Cleavage and Polyadenylation Factors/biosynthesis , Animals , Cytoplasm/genetics , Drosophila , Drosophila Proteins/genetics , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Histones/metabolism , Intranuclear Inclusion Bodies/genetics , Intranuclear Inclusion Bodies/metabolism , Mutation , Nucleoplasmins/genetics , Ribonucleoprotein, U7 Small Nuclear/genetics , S Phase/genetics , mRNA Cleavage and Polyadenylation Factors/genetics
19.
Dev Cell ; 24(6): 623-34, 2013 Mar 25.
Article in English | MEDLINE | ID: mdl-23537633

ABSTRACT

Compartmentalization of RNA biosynthetic factors into nuclear bodies (NBs) is a ubiquitous feature of eukaryotic cells. How NBs initially assemble and ultimately affect gene expression remains unresolved. The histone locus body (HLB) contains factors necessary for replication-coupled histone messenger RNA transcription and processing and associates with histone gene clusters. Using a transgenic assay for ectopic Drosophila HLB assembly, we show that a sequence located between, and transcription from, the divergently transcribed H3-H4 genes nucleates HLB formation and activates other histone genes in the histone gene cluster. In the absence of transcription from the H3-H4 promoter, "proto-HLBs" (containing only a subset of HLB components) form, and the adjacent histone H2a-H2b genes are not expressed. Proto-HLBs also transiently form in mutant embryos with the histone locus deleted. We conclude that HLB assembly occurs through a stepwise process involving stochastic interactions of individual components that localize to a specific sequence in the H3-H4 promoter.


Subject(s)
Drosophila/genetics , Histones/genetics , RNA, Messenger/biosynthesis , Animals , DNA Replication , Drosophila/metabolism , Gene Expression , Histones/metabolism , Promoter Regions, Genetic , RNA, Messenger/genetics , Transcription, Genetic , Transcriptional Activation
20.
Curr Biol ; 22(22): R951-3, 2012 Nov 20.
Article in English | MEDLINE | ID: mdl-23174296

ABSTRACT

A paper in this issue shows that histones H2a and H2b are stored in lipid droplets in Drosophila embryos complexed with the protein Jabba. In Jabba mutant embryos, histones H2a and H2b are degraded but embryos survive by translating stored histone mRNA.


Subject(s)
Drosophila/embryology , Histones/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Mutation , RNA, Messenger/genetics , RNA, Messenger/metabolism
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