Inhibitory Effect of Naked Neural BC1 RNA or BC200 RNA on Eukaryotic in vitro Translation Systems is Reversed by Poly(A)-binding Protein (PABP)

https://doi.org/10.1016/j.jmb.2005.07.049Get rights and content

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.

Introduction

BC1 RNA and BC200 RNA are small non-messenger RNAs,1, 2 transcribed by RNA polymerase III,3 that form ribonucleoprotein particles (RNPs) in the cell.4, 5, 6, 7 Unlike the majority of small stable RNAs, these cytoplasmic RNAs are expressed almost exclusively in neurons, are developmentally regulated, emerge when neurons form connections with other neurons,8 and are transported into dendritic processes.9, 10

BC1 RNA arose by retroposition of tRNAAla and can be found in all rodent species.11 The RNA coding region of the resulting BC1 RNA gene is about twice as long as that of the source gene. This increase can be explained by acquisition of an adenosine-rich region during the retroposition event and recruitment of additional 3′ sequences from the locus of integration up to the point where an RNA polymerase III transcription termination signal was encountered. At the 5′ end, the gene acquired external regulatory elements determining, at least in part, cell type-specific and developmentally regulated expression.3 Furthermore, BC1 RNA served as a master gene for the generation of short interspersed repetitive elements (SINEs); namely, ID elements.12 Although the level of sequence similarity of the 5′ domain of BC1 RNA to tRNAAla is 80%, the tRNA folding of this domain is not apparent anymore and now forms an extended stem–loop structure containing several bulges.13 Close to a dozen proteins have been reported to be components of the BC1 RNP. Most of them are in vitro and/or interpretational artifacts, as exemplified by our own erroneous report on La protein.14 Out of this abundance of candidates, the only certain one is poly(A)-binding protein (PABP).7, 15

Although no homologues have been detected in other mammalian orders, thus far,11 a separate retroposition event led to a small RNA in a different mammalian order whose expression is also prevalent in neurons:10, 16 BC200 RNA is a transcribed monomeric Alu element, itself ancestrally related to signal recognition particle (SRP) RNA. Its phylogenetic distribution is restricted to the Anthropoidea lineage in the order of primates.16, 17, 18 Two of the in vivo associated proteins that bind to BC200 RNA (where the Alu domain of SRP RNA is conserved) are in fact components of the signal recognition particle; namely, SRP9/14.19 The general domain organization of BC200 RNA is similar to that of BC1 RNA. The 5′ domain had been derived from a transcriptionally active monomeric Alu element and represents the translation arrest domain of SRP RNA; an A-rich central region and a unique (non-repetitive) region at its 3′ end follow it. Not surprisingly, PABP is the third reliably identified protein component of the BC200 RNP.7 A detailed account of the evolutionary steps that lead to BC1 and BC200 RNA has been given.16, 20, 21

Several messenger RNAs are also found in dendritic processes of neurons.22, 23 Furthermore, detection of ribosomes and polysomes subjacent to synaptic knobs,24, 25 as well as other components of the translational apparatus has been confirmed.26, 27 Although no definite proof exists, the idea that localized translation modulates synaptic function is widely endorsed.22, 23, 28, 29, 30, 31 If this highly compartmentalized translation of selected mRNAs should fulfil the suggested task, namely delivering protein molecules on site on demand (to the right synapse at the right time) as a molecular basis for synaptic plasticity, then this process hardly could be constitutive and there would be the need to regulate this extrasomatic translation.

The co-distribution of BC1 or BC200 RNA with the extrasomatic translation apparatus/mRNAs and the evolutionary relationship of these small non-mRNAs to RNA species that are intimately involved in translational steps (tRNA and SRP RNA, respectively) and particularly their association with PABP, a regulator of translation renders them viable candidates for participation in such a regulatory role.9, 10, 26, 31 On the other hand, phylogenetic absence of homologues in other mammalian species11 would preclude a central regulatory function in, for example, dendritic translation.

As a first test in order to understand whether BC1 RNA and BC200 RNA may be involved in the process of translational regulation in the cell, we analyzed their influence on the translation of several model mRNAs in vitro in a rabbit reticulocyte system. As a conformation for the obtained in vitro observations, we used RNA cotransfection assays, enabling us to detect influences of cotransfected RNAs on translation of reporter mRNA. We were able to show that, unlike other control RNAs or truncated forms of BC1 RNA or BC200 RNA, these neuronal non-mRNAs exhibited a strong inhibitory effect on translation in vitro and in transfected cell cultures. We demonstrate that one of the crucial domains for this inhibitory effect, at least in an in vitro system or in culture, is the central A-rich domain of the rodent or primate analogues. The inhibition of BC1 RNA or other oligo(A)-containing RNAs could be mitigated by preincubation of the naked RNAs with PABP. In cellular or tissue extracts, BC1 or BC200 RNA have been observed in the form of RNA–protein complexes only. This rules out that a crude mechanism of translational regulation by simply titrating PABP as observed in vitro could be extrapolated to the mechanisms in dendritic processes of neurons. Nevertheless, PABP is an important regulator of translation32 and mediates interaction with other translation factors such as subunits of the eIF4F complex.32, 33, 34, 35 Hence, the respective complex that includes PABP could mediate the potential for a similar but subtler regulatory role for translation.

Section snippets

Naked BC1 RNA and other oligo(A)-containing RNAs inhibit translation in vitro

Addition of BC1 RNA to cell-free rabbit reticulocyte extracts translating a reporter mRNA indicated that BC1 RNA mediated a drastic reduction of protein synthesis.36, 37 The following experiments were designed to expand on this finding. The first experiment examined whether the effect of BC1 RNA on translation was limited to specific mRNAs or whether it was a more general phenomenon. Two sources of bulk mRNA were used as templates for translation in a nuclease-treated, rabbit reticulocyte

Discussion

In this study, we show that neuronally expressed rodent BC1 RNA, its human analog BC200 RNA, as well as other small RNAs containing oligo(A) stretches are able to suppress translation in various systems. B1 and Alu RNAs share with BC200 RNA the Alu domain of the SRP as well as an A-rich region at the 3′ terminus or center of the respective RNAs. BC1 RNA indiscriminately inhibited translation in vitro of most if not all cellular RNAs, independent of their cellular origin (Figure 1). Several

Plasmids and RNA constructs

Plasmid pBCX6075 was linearized by DraI and utilized for in vitro transcription to prepare full-length BC1 RNA (154 nt); RNA representing the 5′ region of BC1 RNA (ID-domain) without the adenosine-rich and unique regions was transcribed from the same plasmid but digested by AvaII (positions 1–65); MaeIII digestion was used to produce the template for in vitro transcription of 5′-ID plus oligo(A)-rich RNA (positions 1–130).

The pPAU plasmid was constructed by PCR-based mutagenesis of pBCX607 as

Acknowledgements

We thank Shirley Tilghman for plasmid pZE.5, Alex Hüttenhofer for plasmids pAG1 and pAF1 as well as for input during initial stages of the investigations, and Tomas Preiss for vectors pOT-CAT and pOT-CAT(A)98. This work was supported, in part, by a grant from the Deutsche Forschungsgemeinschaft (BR 754/2), the German Human Genome Project through the Bundesministerium für Bildung und Forschung (01KW9966), by the Interdisciplinary Centre of Clinical Research (IZKF), Münster (Bro-3/054/04) and a

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      Since PABP influences translation initiation through an interaction with eukaryotic initiation factor 4G (eIF4G) [101], the authors proposed that BC200 and BC1 bind to PABP, forming stable RNPs that can modulate translation initiation [66]. Alternatively, Kondrashov et al. (2005) suggested that BC200 exerts its translational inhibitory effects by acting as a competitor for PABP [102]. This is supported by a subsequent study that observed strong inhibitory effects of naked BC200/BC1 RNAs on translation in reticulocyte lysate and transfected cell systems, while the inclusion of PABP reduced this effect [61].

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    Present addresses: A. V. Kondrashov, Institute of Biomedical and Life Science, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, UK; R. S. Muddashetty, Department of Neuroscience, Rose Kennedy Center for Mental Retardation, Albert Einstein College of Medicine, 1410 Pelham Parkway, Bronx, NY 10461, USA.

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