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The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential forDrosophiladevelopment only when canonical histone gene copy number is reduced, suggesting that coordination between canonicalH3.2and variantH3.3expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reducedH3.2andH3.3gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains thePolycombgene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction inPolycombdosage decreases viability of animals with noH3.3gene copies. Moreover, heterozygousPolycombmutations result in de-repression of the Polycomb target geneUbxand cause ectopic sex combs when either canonical or variantH3gene copy number is also reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variantH3gene copy number falls below a critical threshold.

Polycomb complexes regulate cell type–specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent ( H3.2 K36R ) or replication-independent ( H3.3 K36R ) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined ( H3.3 K36R H3.2 K36R ) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2 K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3 K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

Polycomb complexes regulate cell-type specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants ofDrosophilareplication-dependent(H3.2K36R)or -independent(H3.3K36R)histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined(H3.3K36RH3.2K36R)mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that theH3.2K36Rmutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected inH3.3K36Ranimals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive PREs (Polycomb Response Elements) located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.Short summaryHistone H3.2 and H3.3 K36 residues ensure Hox gene silencing and enable development by different, but synergistic mechanisms.

Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN•pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set8null mutants are inviable, we found that an R634G mutation in the SET domain predicted to ablate catalytic activity resulted in viable adults, suggesting important non-catalytic functions of Set8. Similarly, flies that were engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but they are phenotypically distinct from H4K20R, Set8null, and Set8R634G animals. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP–coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, we discovered a new class of circular non-coding RNAs in metazoans, called tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace native introns of human and fruit fly tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in vivo. Disrupting a conserved base pair in the anticodon-intron helix dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. In contrast, strengthening weak bases in the helix also interferes with splicing and tricRNA production. Furthermore, we identified trans-acting factors important for tricRNA biogenesis, including several known tRNA processing enzymes such as the RtcB ligase and components of the TSEN endonuclease complex. Depletion of these factors inhibits Drosophila tRNA intron circularization. Notably, RtcB is missing from fungal genomes and these organisms normally produce linear tRNA introns. Here, we show that in the presence of ectopic RtcB, yeast lacking the tRNA ligase Rlg1/Trl1 are converted into producing tricRNAs. In summary, our work characterizes the major players in eukaryotic tricRNA biogenesis.

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. However, despite genome-wide correlations between early replication of gene-rich, accessible euchromatin and late replication of gene-poor, inaccessible heterochromatin, a causal relationship between chromatin structure and replication initiation remains elusive. Here, we combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. We found that most pericentric heterochromatin remains late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. We also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome gene expression is up-regulated twofold and replicates earlier in XY males than it does in XX females. We found that H4K16R mutation prevents normal male development and abrogates hyperexpression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. In contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavagein vitro. Moreover, RNAi knockdown of theDrosophilaCLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs)in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

SMN protein levels inversely correlate with the severity of spinal muscular atrophy. The SCFSlmbE3 ligase complex interacts with a degron embedded within the C-terminal self-oligomerization domain of SMN. The findings elucidate a model whereby accessibility of the SMN degron is regulated by self-multimerization., Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

Histone post-translational modifications (PTMs) and differential incorporation of variant and canonical histones into chromatin are central modes of epigenetic regulation. Despite similar protein sequences, histone variants are enriched for different suites of PTMs compared to their canonical counterparts. For example, variant histone H3.3 occurs primarily in transcribed regions and is enriched for “active” histone PTMs like Lys9 acetylation (H3.3K9ac), whereas the canonical histone H3 is enriched for Lys9 methylation (H3K9me), which is found in transcriptionally silent heterochromatin. To determine the functions of K9 modification on variant versus canonical H3, we compared the phenotypes caused by engineering H3.3K9R and H3K9R mutant genotypes in Drosophila melanogaster. Whereas most H3.3K9R and a small number of H3K9R mutant animals are capable of completing development and do not have substantially altered protein coding transcriptomes, all H3.3K9RH3K9R combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data suggest that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, we found that H3.3K9 is methylated at telomeric transposons, and this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, our genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin.

Circular (circ)RNAs have recently become a subject of great biologic interest. It is now clear that they represent a diverse and abundant class of RNAs with regulated expression and evolutionarily conserved functions. There are several mechanisms by which RNA circularization can occur in vivo. Here, we focus on the biogenesis of tRNA intronic circular RNAs (tricRNAs) in archaea and animals, and we detail their use as research tools for orthogonal, directed circRNA expression in vivo.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of theSMN1gene. SMN protein expressed from a paralogous gene,SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in theDrosophilaSMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function, and adult longevity. These disease-related phenotypes are domain-specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages ofDrosophiladevelopment. Due to a large maternal contribution of mRNA and protein,Smnis not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Altogether, these findings illustrate a novelin vivorole for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at critical developmental timepoints that is conserved fromDrosophilato humans.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans., Summary: Using animal models of spinal muscular atrophy, we describe a novel disease mechanism caused by temperature-sensitive protein unfolding/instability of the Tudor domain of SMN.

The presence of introns in both protein-coding and noncoding RNA transcripts is a fascinating phenomenon. It seems counterintuitive that an organism would devote precious time and energy to removing a nucleic acid sequence that will not be present in the final product. Nevertheless, introns (including self-splicing ones) are clearly important components of the basic cellular process of gene expression. Transfer RNA (tRNA) introns have been detected in all three kingdoms of life, and their precise removal is crucial for tRNA function. Of particular interest to this review are the tRNA intronic circular RNAs (tricRNAs) that form during metazoan tRNA splicing. In animal cells, these ultrastable introns form a novel class of noncoding RNA. Here, we summarize established knowledge and describe new findings in the field of tRNA splicing. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA in Disease and Development > RNA in Disease RNA Processing > tRNA Processing.

Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.

Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription., Author summary During development, naïve precursor cells acquire distinct identities through differential regulation of gene expression. The process of cell fate specification is progressive and depends on memory of prior developmental decisions. Maintaining cell identities over time is not dependent on changes in genome sequence. Instead, epigenetic mechanisms propagate information on cell identity by maintaining select sets of genes in either the on or off state. Chemical modifications of histone proteins, which package and organize the genome within cells, are thought to play a central role in epigenetic gene regulation. However, identifying which histone modifications are required for gene regulation, and defining the mechanisms through which they function in the maintenance of cell identity, remains a longstanding research challenge. Here, we focus on the role of histone H3 lysine 27 (H3K27). Modifications of H3K27 are associated with both gene activation and gene silencing (i.e. H3K27 acetylation and methylation, respectively). The histones bearing these modifications are encoded by different histone genes. One set of histone genes is only expressed during cell division, whereas the other set of histone genes is expressed in both dividing and non-dividing cells. Because most cells permanently stop dividing by the end of development, these “replication-independent” histone genes are potentially important for long-term maintenance of cell identity. In this study, we demonstrate that replication-independent H3K27 is required for gene silencing by the Polycomb group of epigenetic regulators. However, despite a strong correlation between replication-independent histones and active genes, we find that replication-independent H3K27 is not required for gene activation. As mutations in replication-independent H3K27 have recently been identified in human cancers, this work may help to inform the mechanisms by which histone mutations contribute to human disease.

Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of twoDrosophilatRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identifiedcis-acting elements required for tricRNA formation in both human and fly cells. We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identifytrans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players inDrosophilatricRNA biogenesis.

A recent publication by Seng et al. in this journal reports the crystallographic structure of refolded, full-length SMN protein and two disease-relevant derivatives thereof. Here, we would like to suggest that at least two of the structures reported in that study are incorrect. We present evidence that one of the associated crystallographic datasets is derived from a crystal of the bacterial Sm-like protein Hfq and that a second dataset is derived from a crystal of the bacterial Gab protein. Both proteins are frequent contaminants of bacterially overexpressed proteins which might have been co-purified during metal affinity chromatography. A third structure presented in the Seng et al. paper cannot be examined further because neither the atomic coordinates, nor the diffraction intensities were made publicly available. The Tudor domain protein SMN has been shown to be a component of the SMN complex, which mediates the assembly of RNA-protein complexes of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). Importantly, this activity is reduced in SMA patients, raising the possibility that the aetiology of SMA is linked to RNA metabolism. Structural studies on diverse components of the SMN complex, including fragments of SMN itself have contributed greatly to our understanding of the cellular UsnRNP assembly machinery. Yet full-length SMN has so far evaded structural elucidation. The Seng et al. study claimed to have closed this gap, but based on the results presented here, the only conclusion that can be drawn is that the Seng et al. study is largely invalid and should be retracted from the literature. published

Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila. Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.

Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.

Gemin4 is a member of the Survival Motor Neuron (SMN) protein complex, which is responsible for the assembly and maturation of Sm-class small nuclear ribonucleoproteins (snRNPs). In metazoa, Sm snRNPs are assembled in the cytoplasm and subsequently imported into the nucleus. We previously showed that the SMN complex is required for snRNP import in vitro, although it remains unclear which specific components direct this process. Here, we report that Gemin4 overexpression drives SMN and the other Gemin proteins from the cytoplasm into the nucleus. Moreover, it disrupts the subnuclear localization of the Cajal body marker protein, coilin, in a dose-dependent manner. We identified three putative nuclear localization signal (NLS) motifs within Gemin4, one of which is necessary and sufficient to direct nuclear import. Overexpression of Gemin4 constructs lacking this NLS sequestered Gemin3 and, to a lesser extent Gemin2, in the cytoplasm but had little effect on the nuclear accumulation of SMN. We also investigated the effects of Gemin4 depletion in the laboratory mouse, Mus musculus. Gemin4 null mice die early in embryonic development, demonstrating that Gemin4 is an essential mammalian protein. When crossed onto a severe SMA mutant background, heterozygous loss of Gemin4 failed to modify the early postnatal mortality phenotype of SMA type I (Smn−/−;SMN2+/+) mice. We conclude that Gemin4 plays an essential role in mammalian snRNP biogenesis, and may facilitate import of the SMN complex (or subunits thereof) into the nucleus., Summary: Gemin4 loss-of-function is recessive lethal in mice, whereas in cell culture its overexpression results in a dominant, gain-of-function relocalization of SMN and other Gemin proteins to the nucleus.

Containing Figures S1 thru S7. (PDF 1904 kb)

Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.

Histone post-translational modifications (PTMs) and differential incorporation of variant and canonical histones into chromatin are central modes of epigenetic regulation. Despite similar protein sequences, histone variants are enriched for different suites of PTMs compared to their canonical counterparts. For example, variant histone H3.3 occurs primarily in transcribed regions and is enriched for “active” histone PTMs like Lys9 acetylation (H3.3K9ac), whereas the canonical histone H3 is enriched for Lys9 methylation (H3K9me), which is found in transcriptionally silent heterochromatin. To determine the functions of K9 modification on variant vs. canonical H3, we compared the phenotypes caused by engineering H3.3K9R and H3K9R mutant genotypes in Drosophila melanogaster. Whereas most H3.3K9R, and a small number of H3K9R, mutant animals are capable of completing development and do not have substantially altered protein-coding transcriptomes, all H3.3K9R H3K9R combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data suggest that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, we found that H3.3K9 is methylated at telomeric transposons and that this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, our genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin.

BackgroundHigh-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis.ResultsThe data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for “enhancer RNAs” (eRNAs) in defining transcriptional programs that are important for animal development.ConclusionsHigh-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.

Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a ‘histone replacement’ system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression. DOI: http://dx.doi.org/10.7554/eLife.23249.001, eLife digest In a single human cell there is enough DNA to stretch over a meter if laid end to end. To fit this DNA inside the cell – which is less than 20 micrometers in diameter – the DNA is tightly wrapped around millions of proteins known as histones, which look like “beads” along a “string” of DNA. These histones can prevent other proteins from binding to DNA and activating specific genes. Therefore, cells use enzymes to chemically modify histones to allow particular stretches of DNA to be unwrapped at specific times. Proteins are made up of building blocks called amino acids. A specific amino acid on histones known as H3K36 is modified in certain sections of DNA that suggest it affects the activities of many genes. However, the precise role of this amino acid remains unclear. Previous studies have tried to investigate this by removing the enzymes that modify it, but these enzymes can also modify many other proteins, making it difficult to know what exactly causes the changes in gene activity. Fruit flies are often used in experiments as models of how genetic processes work in humans and other animals. Like us, fruit flies also package their DNA using histones. To investigate the role of H3K36, Meers et al. generated a mutant fruit fly that has a version of the amino acid that cannot be chemically modified by the normal enzymes. Unexpectedly, the experiments suggest that some changes in gene activity that have been previously reported to be caused by modifying H3K36 might actually be due to other factors. Meers et al. found that H3K36 modifications may instead “mark” certain genes to be more active than they otherwise would be. These findings provide a starting point for understanding exactly how H3K36 regulates gene activity. The next challenge is to refine our understanding of how H3K36 modification affects genes in cancer and other diseases, which may aid the development of new therapies to treat these conditions. DOI: http://dx.doi.org/10.7554/eLife.23249.002

Previous work established Importin-β and Snurportin1 as the vertebrate snRNP import receptor and adaptor proteins, respectively. This study identifies Drosophila Snurportin and shows that it uses an alternative import receptor, Importin7/Moleskin. Moleskin is required for the stability of other snRNP biogenesis factors., Nuclear import is an essential step in small nuclear ribonucleoprotein (snRNP) biogenesis. Snurportin1 (SPN1), the import adaptor, binds to trimethylguanosine (TMG) caps on spliceosomal small nuclear RNAs. Previous studies indicated that vertebrate snRNP import requires importin-β, the transport receptor that binds directly to SPN1. We identify CG42303/snup as the Drosophila orthologue of human snurportin1 (SNUPN). Of interest, the importin-β binding (IBB) domain of SPN1, which is essential for TMG cap–mediated snRNP import in humans, is not well conserved in flies. Consistent with its lack of an IBB domain, we find that Drosophila SNUP (dSNUP) does not interact with Ketel/importin-β. Fruit fly snRNPs also fail to bind Ketel; however, the importin-7 orthologue Moleskin (Msk) physically associates with both dSNUP and spliceosomal snRNPs and localizes to nuclear Cajal bodies. Strikingly, we find that msk-null mutants are depleted of the snRNP assembly factor, survival motor neuron, and the Cajal body marker, coilin. Consistent with a loss of snRNP import function, long-lived msk larvae show an accumulation of TMG cap signal in the cytoplasm. These data indicate that Ketel/importin-β does not play a significant role in Drosophila snRNP import and demonstrate a crucial function for Msk in snRNP biogenesis.

Survival Motor Neuron (SMN) protein localizes to both the nucleus and the cytoplasm. Cytoplasmic SMN is diffusely localized in large oligomeric complexes with core member proteins, called Gemins. Biochemical and cell biological studies have demonstrated that the SMN complex is required for the cytoplasmic assembly and nuclear transport of Sm-class ribonucleoproteins (RNPs). Nuclear SMN accumulates with spliceosomal small nuclear (sn)RNPs in Cajal bodies, sub-domains involved in multiple facets of snRNP maturation. Thus, the SMN complex forms stable associations with both nuclear and cytoplasmic snRNPs, and plays a critical role in their biogenesis. In this review, we focus on potential functions of the nuclear SMN complex, with particular emphasis on its role within the Cajal body.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of SMN isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phospho-degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric, and sequestered when SMN forms higher-order multimers.

Spinal Muscular Atrophy (SMA) is caused by deletion or mutation of theSurvival Motor Neuron 1gene (SMN1)1, but the mechanism whereby reduced levels of SMN protein lead to disease is unknown. SMN functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) and potential splicing defects have been uncovered in various animal models of SMA. We used disruptions inSmnand two additional snRNP biogenesis genes,PhaxandArs2, to classify RNA processing differences as snRNP-dependent orSmngene specific inDrosophila. Although more numerous, the processing changes inArs2mutants were generally distinct from those identified inPhaxandSmnanimals.PhaxandSmnnull mutants exhibited comparable reductions in steady-state snRNA levels, and direct comparison of their transcriptomes uncovered a shared set of alternative splicing changes. Transgenic expression ofPhaxandSmnin the respective mutant backgrounds significantly rescued both snRNA levels as well as alternative splicing. When compared to theSmnwild-type rescue line, three additional disease models (bearing SMA-causing point mutations inSmn) displayed only small-to-indistinguishable differences in snRNA levels and the identified splicing disruptions. Comparison of these intermediate SMA models revealed fewer than 10% shared splicing differences. Instead, the threeSmnpoint mutants displayed common increases in stress responsive transcripts that correlated with phenotypic severity. These findings uncouple organismal viability defects from the general housekeeping function of SMN and suggest that SMN-specific changes in gene expression may be important for understanding how loss of SMN ultimately causes disease.