InteractiveFly: GeneBrief

Nuclear polyadenosine RNA-binding 2: Biological Overview | References


Gene name - Nuclear polyadenosine RNA-binding 2

Synonyms -

Cytological map position - 95F11-95F12

Function - RNA-binding protein

Keywords - poly(A) RNA binding protein - functions in cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP - patterns axon projection in the developing brain

Symbol - Nab2

FlyBase ID: FBgn0028471

Genetic map position - chr3R:24,314,660-24,318,419

NCBI classification - zf-CCCH_2: RNA-binding, Nab2-type zinc finger

Cellular location - cytoplasmic and nuclear



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. This study presents a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14 (Bienkowski, 2017).

RNA binding proteins (RBPs) play important roles in the biogenesis and expression of virtually all types of eukaryotic RNAs, including protein-coding mRNAs. Despite these broad roles, mutations in genes that encode RBPs often lead to tissue-specific disease pathology, particularly within the brain and nervous system. Examples of this link include the fragile X mental retardation protein (FMRP) and the spinal muscular atrophy protein SMN. The prevalence of neurological disorders caused by defects in RBPs likely reflects the enhanced role post-transcriptional mechanisms play in translational control within distal neuronal processes (Bienkowski, 2017).

The ZC3H14 (zinc-finger CysCysCysHis [CCCH]-type 14) gene encodes a ubiquitously expressed RBP that is lost in an inherited form of autosomal, recessive, non-syndromic intellectual disability (Pak, 2011). Patients homozygous for nonsense mutations in ZC3H14 have reduced IQ but lack associated dysmorphic features. Loss of the ubiquitously expressed Drosophila ZC3H14 homolog, dNab2, produces defects in adult viability, motor function, and brain morphology that are fully rescued by neuronal dNab2 re-expression and partially rescued by human ZC3H14 expression (Kelly, 2014, Kelly, 2016, Pak, 2011). These data reveal an important, and evidently conserved, role for human ZC3H14 and fly dNab2 in neurons (Bienkowski, 2017).

ZC3H14 and dNab2 are predominantly localized to the nucleus but are members of a conserved protein family whose founding member, S. cerevisiae Nab2, shuttles between the nucleus and the cytoplasm. ZC3H14 and dNab2 share a domain structure of an N-terminal PWI (proline/tryptophan/isoleucine)-like domain, a nuclear localization sequence, and five well-conserved C-terminal CCCH-type zinc fingers (ZnFs). These ZnF domains bind synthetic polyadenosine RNA probes in vitro, implying that dNab2 and ZC3H14 interact with adenosine-rich tracts in vivo. In support of this hypothesis, ZC3H14 co-localizes with poly(A) mRNA speckles in rodent hippocampal neurons (Pak, 2011), and its loss increases bulk poly(A) tail (PAT) length among RNAs in cultured N2a cells (Kelly, 2014). dNab2 also restricts PAT length in vivo and genetic interactions between dNab2, and components of the polyadenylation machinery (e.g., the PABP poly(A) binding protein and the hiiragi poly(A) polymerase) indicate that altered PAT length may underlie dNab2 mutant phenotypes (Pak, 2011). Altered PAT length can affect multiple steps in RNA metabolism, including turnover and translational efficiency (Eichhorn, 2016, Subtelny, 2014; Bienkowski, 2017 and references therein).

dNab2 plays important roles within the central nervous system (CNS). Pan-neuron dNab2 depletion within the peripheral nervous system (PNS) and CNS replicates almost all phenotypes resulting from zygotic loss of dNab2, while dNab2 depletion from motor neurons does not (Pak, 2011). Moreover, pan-neuron dNab2 depletion impairs short-term memory and disrupts axon projection into the α/β lobes of the mushroom bodies (MBs) (Kelly, 2016), twin neuropil structures in the brain required for associative olfactory learning and memory. In dNab2 mutants, β axons misproject across the brain midline and α axons show a high frequency of branching defects (Kelly, 2016). Selective depletion of dNab2 in Kenyon cells, which give rise to MB α/β axons, is sufficient to phenocopy these dNab2 zygotic defects, and dNab2 re-expression in these cells is sufficient to rescue them (Kelly, 2016). However, there is little evidence of how dNab2 regulates bound RNAs and whether this regulation occurs exclusively in the nucleus, as suggested by the nuclear steady-state localization of dNab2, Nab2, and ZC3H14 (Anderson, 1993, Leung, 2009), or involves a role for dNab2 in cytoplasm (Bienkowski, 2017).

This study describes a genetic screen for dNab2-interacting factors in the Drosophila eye that uncovers physical and functional interactions between dNab2 and the Drosophila ortholog of the FMRP. The FMRP RBP is lost in fragile X syndrome (FXS), the most common genetic cause of intellectual disability. FMRP undergoes nucleocytoplasmic shuttling but is enriched in the cytoplasm at steady state. Cytoplasmic FMRP regulates ~800 polyadenylated neuronal mRNAs, allowing for finely tuned pre- and post-synaptic translation of their encoded proteins. Genetic interactions between dNab2 and the Drosophila FMRP gene (dfmr1) correspond at a molecular level to an RNase-resistant physical association of dNab2 and Drosophila FMRP (dFMRP) proteins in neurons. Within brain neurons, dNab2 and dFMRP co-localize in the soma but are also detected within discrete messenger ribonucleoprotein (mRNP)-like foci distributed along neuronal processes. A corresponding memory defect in dNab2/+,dfmr1/+ trans-heterozygotes indicates that dNab2 may co-regulate a subset of mRNAs bound by dFMRP. dNab2 associates with the dFMRP-regulated mRNA encoding CaMKII (calmodulin-dependent protein kinase-II) and is required for repression of a CaMKII translational reporter in neurons. By contrast, dNab2 does not appear to regulate a second dFMRP-target mRNA encoding Futsch/Map1β, implying that the spectrum of dNab2-regulated mRNAs only partially overlaps with dFMRP. Moreover, this study has found evidence that dFMRP and FMRP restrict PAT length of neuronal mRNAs in a manner similar to dNab2 and ZC3H14. Finally, ZC3H14 was shown to be present in hippocampal axons and dendrites, where it is enriched in ribonucleoprotein (RNP) and 80S ribosomal fractions. Altogether, these data represent a significant advance in understanding dNab2/ZC3H14 by defining a role for these disease-associated RBPs in translational control of neuronal mRNAs that, in Drosophila, occurs in conjunction with the dFMRP protein (Bienkowski, 2017).

This study reports the results of a candidate-based screen for factors that interact genetically with the Drosophila dNab2 gene, which encodes an RBP whose human ortholog is lost in an inherited intellectual disability. Identified interacting genes include components of the translation machinery (PABC1, EF-1α, and eIF-4e) and elements of a pathway centered on the Drosophila ortholog of the FMRP translational repressor (dfmr1 itself, Argonaute-1, Gw182, Rm62, staufen, and Ataxin-2), suggesting that dNab2 functions within the dFMRP pathway. Additional genetic tests support this hypothesis. dfmr1 alleles suppress a rough-eye phenotype caused by transgenic expression of dNab2 in retinal neurons, while dfmr1 alleles enhance a locomotor defect caused by neuronal RNAi of dNab2. Genetic interactions also occur in the CNS, where dfmr1 heterozygosity enhances the frequency of MB α lobe defects in dNab2 mutants. dNab2 heterozygosity suppresses MB α lobe defects in dfmr1 mutants, implying a functional hierarchy in which dNab2 effects are dependent on dFMRP status. The inability of either RBP to rescue phenotypes caused by loss of the other argues for a model in which dNab2 and dFMRP participate in a common mechanism or mechanisms but are not functionally redundant (Bienkowski, 2017).

Genetic interactions between the dNab2 and the dfmr1 genes are paralleled by a dNab2:dFMRP protein complex detected in neurons. This dNab2:dFMRP interaction, which could involve other factors, includes a cytoplasmic pool of dNab2 that partially co-localizes with dFMRP in mRNP-like granules in neuronal processes, suggesting that the two RBPs may associate with some of the same RNAs. dNab2 can interact with and regulate the CaMKII mRNA, a dFMRP target, but is not required to regulate futsch, a second dFMRP target. The finding that trans-heterozygosity for dNab2 and dfmr1 impairs olfactory memory provides additional evidence that dNab2:dFMRP co-regulate some neuronal mRNAs. Finally, this study found that murine ZC3H14 is present in axons and dendrites of murine hippocampal neurons and associates with mRNPs and elements of the translational machinery. FMRP also localizes to dendrites and axons and regulates filopodial dynamics and motility of axonal growth cones. In aggregate, these data significantly advance understanding of the role of dNab2/ZC3H14 proteins in neurons by defining a cytoplasmic pool of these proteins associated with translational control of mRNAs that, in Drosophila, occurs in conjunction with dFMRP (Bienkowski, 2017).

This study highlights the dNab2:dFMRP association but also suggests that dNab2 can function independently of dFMRP. For example, dNab2 and dFMRP are each required for MB αβ┬álobe structure, yet dosage-sensitive interactions between dNab2 and dfmr1 alleles are only evident in α lobes, suggesting that dNab2 and dFMRP may co-regulate RNAs within specific axon branches. In addition, dNab2 selectively regulates CaMKII, but not futsch, and that asymmetry is reflected at the level of the futsch PAT, which is unchanged in dNab2 mutant brains but extended in dfmr1 mutant brains. The failure of dNab2 alleles to alter Futsch protein levels is consistent with their lack of effect on the Futsch-dependent process of NMJ development. Altogether, these data suggest that the futsch mRNA is not a physiological target of dNab2 and that dNab2 only regulates a subset of dFMRP-bound transcripts (Bienkowski, 2017).

dFMRP protein is a well-established translational repressor, but the data reveal a previously unappreciated requirement for dFMRP/FMRP to inhibit mRNA poly(A) tail (PAT) length, which in the case of futsch, is likely to stem from direct binding by dFMRP. This effect on PAT length could simply be a secondary consequence of enhanced futsch translation in dfmr1/Fmr1 mutant cells. However, loss of the cytoplasmic polyadenylation element binding protein (CPEB), which promotes cytoplasmic PAT extension in mammals and flies, rescues FXS phenotypes in Fmr1 knockout mice. One interpretation of this result is that inappropriate PAT elongation contributes to excess translation in FXS, similar to the positive correlation between PAT length and translation observed among germline and embryonic mRNAs. These data thus raise the possibility that altered mRNA polyadenylation may be an unappreciated feature of translational dysregulation in neurons lacking dfmr1/Fmr1 (Bienkowski, 2017).

The dNab2:dFMRP complex suggests that dNab2 may regulate gene expression through its interaction with dFMRP. FMRP inhibits translational initiation, blocks ribosome movement along polyribosome-associated mRNAs, and interacts with elements of the miRNA machinery. The dNab2-sensitive CaMKII-3'UTR GFP sensor is also regulated by the miRNA pathway, and multiple factors involved in miRNA-induced silencing interact genetically with dNab2. The precise role dNab2 plays on bound mRNAs is not clear. PAT elongation induced by dNab2 loss could enhance recruitment of cytoplasmic PABPs that promote translation-coupled circularization of mRNAs. dNab2 and its ortholog ZC3H14 both repress PAT length and may thus indirectly limit the binding of cytoplasmic PABPs to key transcripts. Alternatively, they may directly compete with these PABPs for binding to polyadenosine tails and thus occlude access of other factors involved in translation (Bienkowski, 2017).

Consistent with the role of dNab2 in translational regulation, its ortholog ZC3H14 localizes to axons, dendrites, and dendritic spines in hippocampal neurons and co-sediments with 80S ribosomes. FMRP is primarily associated with polysomes and can inhibit translation by ribosome stalling. The FMRP-target CamKIIα mRNA is enriched in anti-ZC3H14 precipitates, and CaMKIIα levels increase in the hippocampus of Zc3h14Δ13/Δ13 knockout mice compared to control mice, raising the possibility that Drosophila and vertebrate CaMKII mRNAs are conserved targets of dNab2/ZC3H14. The FMRP-related protein Fxr1 co-precipitates with the zinc-finger domain of ZC3H14, suggesting that ZC3H14 may interact with FMRP family members in a manner analogous to dNab2 and dFMRP (Bienkowski, 2017).

Altogether, the data presented in this study provide evidence that dNab2 localizes to both the nucleus and the cytoplasm of Drosophila neuronal processes and that it interacts physically and functionally with the dFMRP protein. Additional data provide evidence of an equivalent pool of cytoplasmic ZC3H14 that interacts with RNP complexes found in the axons and dendrites in the mouse brain. Given the link between FMRP and intellectual disability in humans, these interactions raise the possibility that defects in translational silencing of mRNAs transported to distal sites within neuronal processes contribute to neurodevelopmental and cognitive defects in Drosophila lacking dNab2 or in humans lacking ZC3H14 (Bienkowski, 2017).

The Drosophila ortholog of the Zc3h14 RNA binding protein acts within neurons to pattern axon projection in the developing brain

The dNab2 polyadenosine RNA binding protein is the D. melanogaster ortholog of the vertebrate ZC3H14 protein, which is lost in a form of inherited intellectual disability (ID). Human ZC3H14 can rescue D. melanogaster dNab2 mutant phenotypes when expressed in all neurons of the developing nervous system, suggesting that dNab2/ZC3H14 performs well-conserved roles in neurons. However, the cellular and molecular requirements for dNab2/ZC3H14 in the developing nervous system have not been defined in any organism. This study shows that dNab2 is autonomously required within neurons to pattern axon projection from Kenyon neurons into the mushroom bodies, which are required for associative olfactory learning and memory in insects. Mushroom body axons lacking dNab2 project aberrantly across the brain midline and also show evidence of defective branching. Coupled with the prior finding that ZC3H14 is highly expressed in rodent hippocampal neurons, this requirement for dNab2 in mushroom body neurons suggests that dNab2/ZC3H14 has a conserved role in supporting axon projection and branching. Consistent with this idea, loss of dNab2 impairs short-term memory in a courtship conditioning assay. Taken together these results reveal a cell-autonomous requirement for the dNab2 RNA binding protein in mushroom body development and provide a window into potential neurodevelopmental functions of the human ZC3H14 protein (Kelly, 2016).

The analysis of dNab2 reveals a number of parallels to another RNA binding protein, dFmr1, which is also an ortholog of a protein lost in heritable intellectual disability, FMRP. As with ZC3H14, FMRP is a ubiquitously expressed protein whose loss leads to defects in brain function. Strikingly, dFmr1 mutant flies show adult MB defects very similar to those described here for dNab2 mutant flies, including thinned/missing α lobes and fused β lobes. Human and Drosophila FMRP/dFmr1 are well-established translational repressors, and while the precise molecular role of ZC3H14 and dNab2 have yet to be determined, the role of these proteins in limiting poly(A) tail length (Pak, 2011; Kelly, 2014) suggests that they could impact the fate of mRNAs in the cytoplasm, perhaps via effects upstream of translation. Finally, the dNab2 ortholog ZC3H14 is highly expressed in hippocampal neurons (Pak, 2011), which are also an important site of FMRP action. These similarities between dNab2/ZC3H14 and dFmr1/FMRP are suggestive of potential links between these RNA binding proteins that warrant further investigation (Kelly, 2016).

Given its proposed molecular role as a Pab, dNab2 is likely to support neurodevelopment and memory via effects on the stability and/or translation of neuronal mRNAs. These roles could be linked such that defects in regulation of RNAs supporting axon projection lead to corresponding defects in memory circuits. Alternatively these phenotypes could reflect a requirement for dNab2 in regulating distinct pools of RNAs involved in each process. The current observations that neuronal RNAi-mediated depletion of dNab2 elicits penetrant effects on locomotor behavior (Pak, 2011) and short-term memory, but comparatively mild effects on α/β-lobe structure (approximately 65% of brains affected), suggests these two phenotypes could stem from effects in different cells and perhaps different target RNAs. Indeed some proteins required for courtship memory act in γ-lobe neurons whose structure is unaffected by dNab2 loss, while other proteins are only required in the α/β-lobes. Future studies will need to define dNab2 target RNAs in groups of brain neurons and assess their roles in axon projection and STM phenotypes that arise upon dNab2 loss (Kelly, 2016).

The RNAs responsible for axonal defects in dNab2 mutant Kenyon cells whose projections the α/β MB lobes are as yet undefined. Although dNab2 is localized to the nucleus at steady-state, the budding yeast Nab2 protein shuttles between the cytoplasm and nucleus, presenting the possibility that dNab2 could impact RNA regulatory processes beyond nuclear processing. Studies have implicated a diverse set of molecules in MB development, including the cell-cell adhesion proteins N-cadherin, Down-syndrome cell adhesion molecule (Dscam), and L1CAM, as well as signaling cascades from Ephrin and Wingless/Wnt signals, providing a number of candidate pathways. Coordinated control of these signals during axon outgrowth, bifurcation, and synapse formation likely requires precise temporal and spatial control of mRNA stability, transport, and translation. The dNab2/ZC3H14 Pab restricts poly(A) tail length in vivo. Thus, the required role in MB axon development could stem from effects on one or more transcript(s) involved in axonal projection and branching. Identifying these target RNAs will require functional assays that define dNab2-regulated transcripts in neurons and physical interaction screens that recover transcripts bound by dNab2. The identity of these transcripts will provide important clues as to how dNab2 influences cellular processes in the fly brain. However, equally important will be determining the fate of these RNAs once bound by dNab2, and testing whether dNab2 primarily influences neuronal gene expression by controlling the nuclear export, stability, transport, or translation of cytoplasmic RNAs, even if its role is primarily restricted to controlling poly(A) tail length in the nucleus. This combined analysis of dNab2 targets and how each is regulated by dNab2 will likely shed considerable light on the role of the dNab2/ZC3H14 protein family in brain development and function (Kelly, 2016).

A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. This study provides the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, this study shows that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons (Kelly, 2014).

This study presents the first functional characterization of the mammalian polyadenosine RNA-binding protein, ZC3H14. The data couple evidence from a cultured neuronal cell line with functional studies exploiting a Drosophila model and support a role for ZC3H14 in modulating poly(A) tail length. Significantly, evidence was found that members of this class of zinc finger polyadenosine RNA-binding proteins are functional orthologs in metazoan neurons. Results presented here reveal that the ZC3H14 protein, which is lost in cases of human intellectual disability (Pak, 2011), is required for proper control of poly(A) tail length. Consistent with the observation that human patients lacking ZC3H14 display brain-specific defects, expression of human ZC3H14 solely in the neurons of dNab2 mutant flies is sufficient to rescue both behavioral defects and an underlying molecular defect in poly(A) tail length control. These data identify a conserved cell type, neurons, and a corresponding molecular process, poly(A) tail length control, which are specifically affected by ZC3H14 loss. A key question that remains is to understand the functional consequences of extended poly(A) tails on neuronal development and function, as well as whether the observed neuronal phenotypes are due to changes in specific RNAs or classes of RNAs (Kelly, 2014).

To assess the function of human ZC3H14 in vivo, advantage was taken of a Drosophila model in which functional rescue conferred by tissue-specific expression can be readily assessed. D. melanogaster has been utilized to identify conserved genes and signaling pathways that function in a variety of human diseases ranging from cancer to muscular dystrophy and has proven particularly useful in defining the genetic requirements of higher cognitive function. Genetic and molecular data presented in this study support a model in which the human ZC3H14-iso1 and Drosophila Nab2 proteins function at a common step in RNA processing, and that human ZC3H14 protein can maintain molecular interactions with critical RNAs and proteins in Drosophila neurons, and consequently can elicit similar downstream effects on cellular physiology and development (Kelly, 2014).

Several lines of evidence suggest that the ZC3H14-iso1-3 proteins may have a function distinct from ZC3H14-iso4. First, the protein isoforms localize to different cellular compartments with ZC3H14-iso1-3 localized to the nucleus at steady state, while ZC3H14-iso4 is located in the cytoplasm. Second, expression studies suggest that ZC3H14-iso4 is highly expressed only in the testes. Finally, the most well-characterized ZC3H14 mutation in intellectual disability patients leads to loss of ZC3H14-iso1-3, while ZC3H14-iso4 is likely unaffected. Consistent with the suggestion that the nuclear and cytoplasmic isoforms of ZC3H14 perform distinct functions, this study shows that ZC3H14-iso4 does not replace the function of dNab2. However, a caveat to this conclusion is that levels of transgenic ZC3H14-iso4 attained are not comparable to those obtained for transgenic expression of ZC3H14-iso1. Furthermore, the transgenic proteins are both N-terminally epitope-tagged which could interfere with protein function. Thus, the results suggest that ZC3H14-iso4 is not a functional ortholog of dNab2 but further studies would be required to directly address this point (Kelly, 2014).

This analysis provides evidence that rescue of both organismal and behavioral defects in the dNab2 mutant flies correlates with a rescue of a molecular defect in polyadenylation. However, this work does not directly demonstrate that altered polyadenylation underlies the Drosophila phenotypes. Given the interdependent nature of transcription, RNA processing, export, and translation, dNab2/ZC3H14 could play direct roles in functions other than control of poly(A) tail length. Indeed, recent work has defined roles for the S. pombe Nab2 protein in regulated RNA turnover and other studies identified the corresponding C. elegans protein, termed Sut-2, as a suppressor of a Tau toxicity model. Understanding the precise molecular functions of RNA-binding proteins, as well as defining their target RNAs, is a significant challenge and a major focus of ongoing studies of dNab2/ZC3H14 (Kelly, 2014).

Although most mRNA transcripts are polyadenylated at the 3'-end, it is infered from this analysis that the shared molecular properties of the Nab2/ZC3H14 nuclear poly(A)-binding proteins are nonetheless more critical in neurons than in other cell types. This inference is congruent with the fact that mutation of ZC3H14 in humans causes nonsyndromic intellectual disability (Pak, 2011), a form of disease where brain function is impaired without other detectable symptoms. The enhanced requirement for dNab2/ZC3H14 in neurons relative to other cell types also parallels an existing body of evidence showing that regulated polyadenylation is an important mechanism that guides the localized translation of mRNAs involved in synaptic development and plasticity. Altered polyadenylation, as occurs in Drosophila and mammalian neurons lacking dNab2/ZC3H14, could perturb or override this cytoplasmic coupling between poly(A) tail length and translation and thus alter expression of RNAs that are subject to both temporal and spatial control (Kelly, 2014).

The present study demonstrating that human ZC3H14 is a functional ortholog of Drosophila Nab2 provides a validated genetic model that can be used to understand how defects in otherwise ubiquitously expressed RNA-binding proteins give rise to neuron-specific dysfunction. ZC3H14 joins a growing class of RNA-binding proteins that when disrupted or silenced result in neuron-specific diseases. Other members of this class of proteins include the Fragile-X Syndrome (FXS) protein, FMRP, and the spinal muscular atrophy (SMA) protein, SMN1. The specific dependence of neurons on Nab2/ZC3H14 fits well with an emerging model that the 3' UTRs of neuronal RNAs are subject to more extensive regulation than their non-neuronal counterparts. Future studies investigating the precise molecular role of ZC3H14 and the neuronal target RNAs that it binds and regulates will be critical in defining how defects in Nab2/ZC3H14-dependent post-transcriptional regulation of gene expression contribute to neuronal dysfunction (Kelly, 2014).

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans

A human intellectual disability disease locus on chromosome 14q31.3 corresponds to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. ZC3H14 mRNA transcripts were identified in the human central nervous system, and it was found that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder (Pak, 2011).

In an effort to better understand the molecular and cellular processes that underlie normal brain function, this study has sought to identify mutations that lead to ID in the human population. This study identified mutations in the human ZC3H14 gene in human patients with NS-ARID and created a tractable genetic model that recapitulates key phenotypic elements of the human disease. The Drosophila ZC3H14 ortholog dNab2 regulates RNA poly(A) tail length and that loss of dNab2 leads to extended RNA poly(A) tails. The effect of dNab2 on RNA poly(A) tail length coupled with neuronal-specific behavioral phenotypes seen in dNab2 mutant flies and ZC3H14-associated NS-ARID patients provide evidence that dNab2-mediated control of RNA poly(A) tail length is required for normal neuronal function (Pak, 2011).

Loss of dNab2 in Drosophila or NAB2 in budding yeast causes an increase in bulk RNA poly(A) tail length, but the mechanism by which these hyperadenylated mRNAs contribute to neuronal dysfunction or possibly, to human disease is not established. Likely consequences of hyperadenylated mRNAs could include altered transcript stability, titration of critical poly(A) RNA binding proteins, and/or bypass of cytoplasmic polyadenylation necessary for activity-dependent translation of neuronal mRNAs. Individually or combined, these defects could disrupt spatiotemporal control of gene expression needed for development of the nervous system and higher-order brain function. Thus, it is speculated that ZC3H14/dNab2 could play critical roles in neurons such as ensuring that transcripts are properly targeted to sites of localized translation. This hypothesis is consistent with a report that budding yeast Nab2 aids in targeting transcripts to the bud site. Alternatively, ZC3H14 and dNab2 may regulate a set of mRNAs that play key roles in neurons, such that ZC3H14/dNab2 loss disproportionately affects this cell type. These mechanisms could explain why mutation of ubiquitously expressed posttranscriptional regulatory factors such as dNab2 and ZC3H14 leads to neuronal defects in flies and more critically, to NS-ARID in humans (Pak, 2011).

Although the NAB2 and dNab2 genes are essential, loss of the corresponding forms of the ZC3H14 protein (isoforms 1-3) in humans seems to selectively impair brain function, because patients display nonsyndromic intellectual disability. At this early stage of investigation, it is unclear whether ZC3H14 is simply not essential in humans or whether the remaining cytoplasmic isoform of the protein, isoform 4, suffices in all tissues except the brain. Alternatively, it cannot be ruled out that a protein that is functionally redundant with ZC3H14 exists; however, the human genome does not encode any apparent sequence orthologs of ZC3H14. Because isoform 4 is only expressed in mammals, future studies exploiting mammalian model systems will be required to address the functional requirements for specific isoforms of ZC3H14 as they relate to human intellectual disability (Pak, 2011).

The identification of ZC3H14 mutations in NS-ARID places ZC3H14/dNab2 among several other RNA binding proteins implicated in human diseases that impact neural function. However, this study identifies a direct link between a poly(A) RNA binding protein and a human brain disorder and thus provides insight into the molecular basis of intellectual disability and brain function (Pak, 2011).


Functions of Nab2 orthologs in other species

The polyadenosine RNA-binding protein ZC3H14 interacts with the THO complex and coordinately regulates the processing of neuronal transcripts

The polyadenosine RNA-binding protein ZC3H14 is important in RNA processing. Although ZC3H14 is ubiquitously expressed, mutation of the ZC3H14 gene causes a non-syndromic form of intellectual disability. This study examine the function of ZC3H14 in the brain by identifying ZC3H14-interacting proteins using unbiased mass spectrometry. Through this analysis, physical interactions were identified between ZC3H14 and multiple RNA processing factors. Notably, proteins that comprise the THO nuclear export complex were among the most enriched proteins. ZC3H14 was found to physically interact with THO components, and these proteins are required for proper RNA processing, as loss of ZC3H14 or THO components leads to extended bulk poly(A) tail length. Furthermore, the transcripts Atp5g1 and Psd95 were identified as shared RNA targets of ZC3H14 and the THO complex. These data suggest that ZC3H14 and the THO complex are important for proper processing of Atp5g1 and Psd95 RNA, as depletion of ZC3H14 or THO components leads to decreased steady-state levels of each mature transcript accompanied by accumulation of Atp5g1 and Psd95 pre-mRNA in the cytoplasm. Taken together, this work provides the first unbiased identification of nuclear ZC3H14-interacting proteins from the brain and links the functions of ZC3H14 and the THO complex in the processing of RNA (Morris, 2018).

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. Mutations have been identified in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, a mouse was generated in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Deltaex13/Deltaex13) leading to a truncated ZC3H14 protein. As in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Deltaex13/Deltaex13mice, this study provides the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Deltaex13/Deltaex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Deltaex13/Deltaex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Deltaex13/Deltaex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo (Rha, 2017).

The evolutionarily-conserved polyadenosine RNA binding protein, Nab2, cooperates with splicing machinery to regulate the fate of pre-mRNA

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, this study investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo. This analysis was extended to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. The interaction between ZC3H14 and U2AF2/U2AF(65) was validated. Taking all the findings into consideration, a model is presented where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation (Soucek, 2016).

Recognition of polyadenosine RNA by the zinc finger domain of nuclear poly(A) RNA-binding protein 2 (Nab2) is required for correct mRNA 3'-end formation

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, this study defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. Genetic interactions were demonstrated between mutant nab2 alleles and mutant alleles of the mRNA 3'-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length (Kelly, 2010).

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2, 3, and 3 short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain (Leung, 2009).


REFERENCES

Search PubMed for articles about Drosophila Nab2

Anderson, J. T., Wilson, S. M., Datar, K. V. and Swanson, M. S. (1993). NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability. Mol Cell Biol 13(5): 2730-2741. PubMed ID: 8474438

Bienkowski, R. S., Banerjee, A., Rounds, J. C., Rha, J., Omotade, O. F., Gross, C., Morris, K. J., Leung, S. W., Pak, C., Jones, S. K., Santoro, M. R., Warren, S. T., Zheng, J. Q., Bassell, G. J., Corbett, A. H. and Moberg, K. H. (2017). The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile X Protein ortholog in Drosophila neurons. Cell Rep 20(6): 1372-1384. PubMed ID: 28793261

Eichhorn, S. W., Subtelny, A. O., Kronja, I., Kwasnieski, J. C., Orr-Weaver, T. L. and Bartel, D. P. (2016). mRNA poly(A)-tail changes specified by deadenylation broadly reshape translation in Drosophila oocytes and early embryos. Elife 5. PubMed ID: 27474798

Kelly, S. M., Leung, S. W., Apponi, L. H., Bramley, A. M., Tran, E. J., Chekanova, J. A., Wente, S. R. and Corbett, A. H. (2010). Recognition of polyadenosine RNA by the zinc finger domain of nuclear poly(A) RNA-binding protein 2 (Nab2) is required for correct mRNA 3'-end formation. J Biol Chem 285(34): 26022-26032. PubMed ID: 20554526

Kelly, S., Pak, C., Garshasbi, M., Kuss, A., Corbett, A. H. and Moberg, K. (2012). New kid on the ID block: neural functions of the Nab2/ZC3H14 class of Cys(3)His tandem zinc-finger polyadenosine RNA binding proteins. RNA Biol 9(5): 555-562. PubMed ID: 22614829

Kelly, S. M., Leung, S. W., Pak, C., Banerjee, A., Moberg, K. H. and Corbett, A. H. (2014). A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length. RNA 20(5): 681-688. PubMed ID: 24671764

Kelly, S. M., Bienkowski, R., Banerjee, A., Melicharek, D. J., Brewer, Z. A., Marenda, D. R., Corbett, A. H. and Moberg, K. H. (2016). The Drosophila ortholog of the Zc3h14 RNA binding protein acts within neurons to pattern axon projection in the developing brain. Dev Neurobiol 76(1): 93-106. PubMed ID: 25980665

Leung, S. W., Apponi, L. H., Cornejo, O. E., Kitchen, C. M., Valentini, S. R., Pavlath, G. K., Dunham, C. M. and Corbett, A. H. (2009). Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein. Gene 439(1-2): 71-78. PubMed ID: 19303045

Morris, K. J. and Corbett, A. H. (2018). The polyadenosine RNA-binding protein ZC3H14 interacts with the THO complex and coordinately regulates the processing of neuronal transcripts. Nucleic Acids Res 46(13): 6561-6575. PubMed ID: 29912477

Pak, C., Garshasbi, M., Kahrizi, K., Gross, C., Apponi, L. H., Noto, J. J., Kelly, S. M., Leung, S. W., Tzschach, A., Behjati, F., Abedini, S. S., Mohseni, M., Jensen, L. R., Hu, H., Huang, B., Stahley, S. N., Liu, G., Williams, K. R., Burdick, S., Feng, Y., Sanyal, S., Bassell, G. J., Ropers, H. H., Najmabadi, H., Corbett, A. H., Moberg, K. H. and Kuss, A. W. (2011). Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans. Proc Natl Acad Sci U S A 108(30): 12390-12395. PubMed ID: 21734151

Rha, J., Jones, S. K., Fidler, J., Banerjee, A., Leung, S. W., Morris, K. J., Wong, J. C., Inglis, G. A. S., Shapiro, L., Deng, Q., Cutler, A. A., Hanif, A. M., Pardue, M. T., Schaffer, A., Seyfried, N. T., Moberg, K. H., Bassell, G. J., Escayg, A., Garcia, P. S. and Corbett, A. H. (2017). The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice. Hum Mol Genet 26(19): 3663-3681. PubMed ID: 28666327

Soucek, S., Zeng, Y., Bellur, D. L., Bergkessel, M., Morris, K. J., Deng, Q., Duong, D., Seyfried, N. T., Guthrie, C., Staley, J. P., Fasken, M. B. and Corbett, A. H. (2016). The evolutionarily-conserved polyadenosine RNA binding protein, Nab2, cooperates with splicing machinery to regulate the fate of pre-mRNA. Mol Cell Biol. PubMed ID: 27528618

Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H. and Bartel, D. P. (2014). Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508(7494): 66-71. PubMed ID: 24476825


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date revised: 20 October 2018

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