Interactions of noncanonical motifs with hnRNP A2 promote activity-dependent RNA transport in neurons

A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell’s activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca²⁺ levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca²⁺ through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca²⁺-dependent RNA–protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.     

Spatial code recognition in neuronal RNA targeting: role of RNA-hnRNP A2 interactions

In neurons, regulation of gene expression occurs in part through translational control at the synapse. A fundamental requirement for such local control is the targeted delivery of select neuronal mRNAs and regulatory RNAs to distal dendritic sites. The nature of spatial RNA destination codes, and the mechanism by which they are interpreted for dendritic delivery, remain poorly understood. We find here that in a key dendritic RNA transport pathway (exemplified by BC1 RNA, a dendritic regulatory RNA, and protein kinase M ζ [PKMζ] mRNA, a dendritic mRNA), noncanonical purine•purine nucleotide interactions are functional determinants of RNA targeting motifs. These motifs are specifically recognized by heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), a trans-acting factor required for dendritic delivery. Binding to hnRNP A2 and ensuing dendritic delivery are effectively competed by RNAs with CGG triplet repeat expansions. CGG repeats, when expanded in the 5′ untranslated region of fragile X mental retardation 1 (FMR1) mRNA, cause fragile X–associated tremor/ataxia syndrome. The data suggest that cellular dysregulation observed in the presence of CGG repeat RNA may result from molecular competition in neuronal RNA transport pathways.     

Systems analysis of RNA trafficking in neural cells

In neural cells, certain RNAs are targeted to dendrites by a specific RNA trafficking pathway, termed the A2 pathway, mediated by the trans-acting trafficking factor, heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which binds to an 11 nucleotide cis-acting trafficking sequence, termed the hnRNP A2 response element (A2RE). RNAs containing A2RE-like sequences are recognized by hnRNP A2 in the nucleus and exported to the cytoplasm where they assemble into trafficking intermediates, termed granules, which also contain components of the translation machinery and molecular motors (cytoplasmic dynein and conventional kinesin). RNA granules move along microtubules to the cell periphery where they become localized and where the encoded protein is translated. Intracellular trafficking of RNA molecules by the A2 pathway is mediated by a complex system consisting of five different subsystems, approximately 35 different molecules and approximately 45 different molecular interactions. Specificity in the A2 pathway is provided by specific interactions of hnRNP A2 with different molecular partners in different subsystems. Polarity of RNA trafficking is controlled by transitions of trafficking intermediates between different subsystems. Comprehensive understanding of the A2 RNA trafficking pathway will require quantitative analysis of concentrations and diffusion constants for each of the different molecules, on rates and off rates for each of the different interactions, relevant conditional operators controlling specific interactions, and interactions of different subsystems. Once the necessary quantitative data are available, mathematical models for the different RNA trafficking subsystems can be developed using computational platforms such as the 'Virtual Cell'. Here we describe how each of the subsystems in the A2 system functions and how the different subsystems interact to regulate RNA trafficking.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.     

Analysis of Subcellularly Localized mRNAs Using in Situ Hybridization,mRNA Amplification,and Expression Profiling

Targeting of mRNAs to distinct subcellular regions occurs in all polarized cells. The mechanisms by which RNA transport occurs are poorly understood. With the advent of RNA amplification methodologies and expression profiling it is now possible to catalogue the RNAs that are targeted to particular subcellular regions. In particular, neurons are polarized cells in which dendrites receive signals from presynaptic neurons. Upon stimulation (information receipt) the dendrite processes the information such that an immediate dendritic response is generated as well as a longer-term somatic response. The integrated cellular response results in a signal that can be propagated through the axon to the next post-synaptic neuron. Much previous work has shown that mRNAs can be localized in dendrites and that local translation in dendrites can occur. In this chapter the methods for analysis of RNAs that are localized to dendrites are reviewed and a partial list of dendritically localized RNAs is presented. This information may be useful in identifying RNA regulatory regions that are responsible for specifying rate of RNA transport and the dendritic sites at which targeted RNAs dock so that they can be translated.     

Poster Sessions CP05: Intracellular Transport and Vesicle Trafficking. MBP mRNA trafficking factor HNRNP A2 interacts with microtubules binding protein TOG

HnRNP A2 is an RNA trafficking protein that binds to a specific cis‐acting RNA trafficking element (A2RE) in myelin basic protein RNA and other transported RNAs. A2RE/hnRNPA2 determinants mediate several different steps in RNA trafficking including granule assembly, transport to the plus ends of microtubules and translational activation. A yeast two hybrid screen designed to identify proteins that interact with hnRNP A2 selected a clone corresponding to the carboxyl terminal portion of TOG (tumor overexpressed gene), a microtubule‐associated protein that regulates microtubule dynamics. Co‐immunostaining of oligodendrocytes with antibody to hnRNPA2 and TOG revealed extensive colocalization of TOG with hnRNP A2 granules in the dendrites. A small population of hnRNP A2 granules lacked TOG and some regions of TOG staining lacked hnRNP A2. In oligodendrocytes injected with fluorescent A2RE RNA and stained for TOG, granules containing fluorescent RNA colocalized with TOG. Co‐injection of anti‐TOG antibody with fluorescent A2RE RNA decreased colocalization with TOG and increased transport of the injected RNA. These observations suggest that molecular interaction between hnRNP A2 and TOG serves to anchor A2RE mRNAs/hnRNPA2 granules at plus ends of microtubules. Acknowledgements: Supported by NIH NS19943 (EB) and NS15190 (JHC), and NMSS RG2843 (EB).     

Endothelin-B receptor mediates effect of intravitreal endothelin on mitochondria-associated anterograde axonal transport

HnRNP A2 is an RNA trafficking protein that binds to a specific cis -acting RNA trafficking element (A2RE) in myelin basic protein RNA and other transported RNAs. A2RE/hnRNPA2 determinants mediate several different steps in RNA trafficking including granule assembly, transport to the plus ends of microtubules and translational activation. A yeast two hybrid screen designed to identify proteins that interact with hnRNP A2 selected a clone corresponding to the carboxyl terminal portion of TOG (tumor overexpressed gene), a microtubule-associated protein that regulates microtubule dynamics. Co-immunostaining of oligodendrocytes with antibody to hnRNPA2 and TOG revealed extensive colocalization of TOG with hnRNP A2 granules in the dendrites. A small population of hnRNP A2 granules lacked TOG and some regions of TOG staining lacked hnRNP A2. In oligodendrocytes injected with fluorescent A2RE RNA and stained for TOG, granules containing fluorescent RNA colocalized with TOG. Co-injection of anti-TOG antibody with fluorescent A2RE RNA decreased colocalization with TOG and increased transport of the injected RNA. These observations suggest that molecular interaction between hnRNP A2 and TOG serves to anchor A2RE mRNAs/hnRNPA2 granules at plus ends of microtubules.
Acknowledgements:   Supported by NIH NS19943 (EB) and NS15190 (JHC), and NMSS RG2843 (EB).     

Conditional Knockout of Tumor Overexpressed Gene in Mouse Neurons Affects RNA Granule Assembly,Granule Translation,LTP and Short Term Habituation

In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

The cis-acting RNA trafficking signal from myelin basic protein mRNA and its cognate trans-acting ligand hnRNP A2 enhance cap-dependent translation.

The 21 nucleotide RNA trafficking signal (RTS), originally identified in myelin basic protein mRNA, but also found in a variety of other localized RNAs, is necessary and sufficient for transport of RNA along microtubules in oligodendrocytes. The RTS binds specifically to the RNA binding protein, hnRNP A2. Together, the RTS and hnRNP A2 comprise cis/trans determinants for several steps in the RNA trafficking pathway. Here we show that insertion of the RTS into green fluorescent protein (GFP) RNA enhances translation without affecting stability of microinjected RNA. In dicistronic RNA, the RTS enhances cap-dependent translation without affecting internal ribosome entry site (IRES)-dependent translation. The translation enhancer function of the RTS is position, copy number, and cell type independent, hnRNP A2 dependent, and saturable with increasing amounts of injected RNA. This represents one of the first specific translation enhancer elements identified in a mammalian system.     

SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV

DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2), is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV). However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1), the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2), CLASSY1 (CLSY1), and RNA-directed DNA methylation 4 (RDM4), suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1), was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.     

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.     

A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.