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. 2012 Jan 6;287(2):1322-34.
doi: 10.1074/jbc.M111.315291. Epub 2011 Nov 29.

Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells

Hatmone Miroci  1 , Claudia Schob, Stefan Kindler, Janin Ölschläger-Schütt, Susanne Fehr, Tassilo Jungenitz, Stephan W Schwarzacher, Claudia Bagni, Evita Mohr
Affiliations

Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells

Hatmone Miroci et al. J Biol Chem. .

Abstract

The poly(A)-binding protein (PABP), a key component of different ribonucleoprotein complexes, plays a crucial role in the control of mRNA translation rates, stability, and subcellular targeting. In this study we identify RING zinc finger protein Makorin 1 (MKRN1), a bona fide RNA-binding protein, as a binding partner of PABP that interacts with PABP in an RNA-independent manner. In rat brain, a so far uncharacterized short MKRN1 isoform, MKRN1-short, predominates and is detected in forebrain nerve cells. In neuronal dendrites, MKRN1-short co-localizes with PABP in granule-like structures, which are morphological correlates of sites of mRNA metabolism. Moreover, in primary rat neurons MKRN1-short associates with dendritically localized mRNAs. When tethered to a reporter mRNA, MKRN1-short significantly enhances reporter protein synthesis. Furthermore, after induction of synaptic plasticity via electrical stimulation of the perforant path in vivo, MKRN1-short specifically accumulates in the activated dendritic lamina, the middle molecular layer of the hippocampal dentate gyrus. Collectively, these data indicate that in mammalian neurons MKRN1-short interacts with PABP to locally control the translation of dendritic mRNAs at synapses.

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Figures

FIGURE 1.
FIGURE 1.
PABP interacts with the RING zinc finger protein MKRN1 in an RNA-independent manner. A, the upper panel shows the genomic organization of the human MKRN1 gene, which consists of exons 1–8. The mRNAs encoding MKRN1-long and MKRN1-short, respectively, are generated by alternative splicing and differential polyadenylation (lower panel). Consequently, MKRN1-long and -short mRNAs possess different 3′-untranslated regions. Poly(A), polyadenylation signal; stop, stop codon. B, MKRN1-long is a modular protein consisting of four zinc fingers (ZF), a MKRN-type zinc finger (MTZF), and a RING finger domain (RF). C, MKRN1-short is C-terminally truncated and lacks ZF4 and the last six amino acids of the RING finger domain including two essential cysteine residues (RFΔCC). D, FLAG-PABP and/or T7-MKRN1-short were expressed in HEK-293 cells. The control shows non-transfected cells. Protein extracts were subjected to immunoprecipitation with either anti-FLAG-agarose (lanes 2, 3, and 7) or anti-T7-agarose (lane 5) in the absence (−) or presence (+) of RNase A. The inputs (I) are shown in lanes 1, 4, and 6. Western blots were probed with anti-PABP- and anti-MKRN1-short antibodies, respectively. The non-transfected control (lanes 6 and 7) shows no unspecific binding of PABP to anti-FLAG-agarose. RNAs extracted before (lane 1) and after immunoprecipitation (lanes 2 and 3) were resolved by agarose gel electrophoresis followed by ethidium bromide staining (lower panel, left). I, input; E, protein eluted from anti-FLAG- or anti-T7-agarose. The positions of molecular size marker proteins (in kDa) and the 18 S and 28 S ribosomal RNAs are indicated on the left.
FIGURE 2.
FIGURE 2.
Identification of the PABP binding site of MKRN1-short. A, shown is a schematic representation of recombinant EGFP-MKRN1-short fusion proteins (encoded by M1-M8) that were transiently expressed in HEK-293 cells together with FLAG-PABP. B–F, protein extracts were subjected to immunoprecipitation with anti-FLAG-agarose followed by SDS-PAGE and Western blot (WB) analyses using anti-GFP antibodies for detection of EGFP-MKRN1 deletion mutants (B–D) or anti-PABP antibodies (E and F). B and E: inputs; C and F, eluted immunoprecipitates; D, supernatant after immunoprecipitation. The positions of molecular size marker proteins (in kDa) are indicated on the right. E, MTZF, Makorin-type zinc-finger; RFΔCC, truncated RING finger domain.
FIGURE 3.
FIGURE 3.
MKRN1-short contains a PAM2-like motif that mediates its interaction with PABP. A, shown is sequence alignment of GW182 family DUF domains and PAM2 motifs of the PABP-binding proteins PAIP1, PAIP2, and ATXN2 with the PABP-interaction motif in MKRN1-short. Strictly conserved residues are highlighted in turquoise. Residues conserved within the DUF and PAM2 domains are shown in magenta and yellow, respectively. The second most critical residue, leucine, within the PAM2 domain, is highlighted in red. Numbers refer to the amino acid position within the respective proteins. B, recombinant EGFP (lanes 1 and 3) or the MKRN1-short PAM2-like domain (amino acids 161–193) fused to EGFP (EGFP-PAM2MKRN; lanes 2 and 4) were transiently expressed in HEK-293 cells. Protein extracts were subjected to immunoprecipitation with GFP-Trap®_A beads followed by SDS-PAGE and Western blot (WB) analyses using anti-GFP antibodies for detection of EGFP and EGFP-PAM2MKRN (upper panel) or anti-PABP antibodies for detection of endogenous PABP (lower panel), respectively. The positions of molecular size marker proteins (in kDa) are indicated on the right. Dm, Drosophila melanogaster; Hs, Homo sapiens; TNRC, trinucleotide repeat containing gene protein.
FIGURE 4.
FIGURE 4.
MKRN1-short binds to several domains of PABP. A, shown is a schematic representation of recombinant EGFP-PABP fusion proteins (encoded by P1-P5) that were transiently expressed in HEK-293 cells together with T7-MKRN1-short. WB, Western blot. B–F, protein extracts were subjected to immunoprecipitation with GFP-Trap®_A followed by SDS-PAGE and Western blot analyses using anti-GFP antibodies for detection of EGFP-PABP (B and C) or anti-T7 antibodies (D–F) for detection of recombinant MKRN1-short. B and D, inputs; C and E, eluted immunoprecipitates; F, supernatant after immunoprecipitation. The positions of molecular size marker proteins (in kDa) are indicated on the right. aa, amino acids.
FIGURE 5.
FIGURE 5.
Characterization of MKRN1 expression in rat brain at the mRNA and protein level. A, poly(A)-RNAs from various rat tissues were hybridized with a 32P-labeled MKRN1 probe that detects both MKRN1-long (3.2 kb) and MKRN1-short (2 kb) transcripts. The asterisk denotes a third transcript variant of 0.75 kb. The positions of marker RNAs (in kb) are indicated on the left. B, RT-PCR with rat hippocampal RNA was performed to confirm the expression of MKRN1-short (lane 1, 554 bp) and MKRN1-long (lane 4, 407 bp) in this brain region. The primers used for the amplification reactions (open arrows) are schematically shown in the lower part of this panel. As controls, PCRs were done in the absence of template (lanes 2 and 5) as well as with non-reverse transcribed RNA (lanes 3 and 6). PCR products were resolved by agarose gel electrophoresis along with a 100-bp ladder marker (left) followed by ethidium bromide staining. C, rat hippocampal proteins were probed with rabbit anti-MKRN1 antiserum (lane 1). Human T7-tagged MKRN1-short (lane 2) and MKRN1-long (lane 3) expressed in HEK-293 cells were run in parallel on the same gel. Antibodies raised against human MKRN1-short recognize recombinant myc-His-tagged rat MKRN1-short (lane 5). Non-transfected control extract is shown in lane 4. D, MKRN1-long is only efficiently expressed in HEK-293 cells if proteasomal degradation is inhibited by the addition of MG-132 (final concentration, 10 μm). The compound was added 1, 2, or 4 h (lanes 2–4) before protein extraction. Bands marked by asterisks most likely correspond to ubiquitinated forms of MKRN1-long. MKRN1-short lacks autoubiquitination properties, probably due to the C-terminal deletion of six amino acids of its RING finger domain. Destabilization of the short MKRN1 variant is not seen if cells are grown in the absence of MG-132 (lane 5). The addition of the compound for various periods of time does not alter the steady state concentration of MKRN1-short (lanes 6–8). The positions of molecular size marker proteins (in kDa) are indicated on the left. WB, Western blot.
FIGURE 6.
FIGURE 6.
MKRN1 is located in the cell nuclei and in the cytoplasm and localizes to neurites of nerve cells where it is partially co-localized with PABP. A and B, HeLa cells transfected with an Myc-tagged MKRN1-short encoding construct were immunostained with a monoclonal mouse anti-myc antibody. Recombinant protein is detected in the cell nuclei as well as in the cytoplasm. C and D, in vitro cultured rat hippocampal neurons transfected with Myc-tagged MKRN1-short encoding construct were immunostained with rabbit anti-MKRN1-short antiserum. Recombinant protein is detected in the cell body as well as in the dendritic tree. A neurite devoid of MAP2-staining (shown in D), a marker of the dendritic cytoskeleton, is also decorated by antibodies (C open arrowheads). E and F, sagittal rat hippocampal section (dentate gyrus) stained with rabbit anti-MKRN1-short antiserum is shown. Strongest immunoreactivity is seen in the granule cell body layer (arrow). G and H, higher power magnification of granule cells shown in E reveals MKRN1-staining in the nuclei and in the cytoplasm. Staining proceeds to the proximal parts of dendrites. I–K, in vitro cultured rat hippocampal neurons transfected with a T7-tagged MKRN1-short encoding construct were immunostained with mouse anti-T7 and rabbit anti-PABP antibodies. L, shown is higher power magnification of the dendritic segment encircled by the white rectangle in panel K. The open arrowheads denote examples of yellow-stained regions of MKRN1-short and PABP colocalization.
FIGURE 7.
FIGURE 7.
MKRN1-short stimulates translation in nerve cells. The eukaryotic expression vector pinFiRein-boxB16B was co-transfected with vectors encoding fusion proteins consisting of an N-terminal N22 peptide and either hMKRN1-short, hMKRN1-Δ487–535, hDDX6, or rShank3–1-299 into dispersed cortical neurons at 7 DIV. The empty vector N22-FLAG3 served as a control (for details see “Experimental Procedures”). A, dual luciferase assays were performed at 9 DIV. The relative levels of PhoLuc/RenLuc proteins are shown (in arbitrary units). B, RNAs from transfected neurons were prepared on 9 DIV, transcribed into cDNAs, and subjected to real-time PCR analyses using Pho/Luc- and RenLuc-specific TaqMan assays. The relative levels of PhoLuc/RenLuc mRNAs are shown (in arbitrary units). Bars represent S.E. Statistical analyses were done using Student's t test (**, p < 0.01; ***, p < 0.001, n.s., not significant). C and D, ribosomes/polysomes from adult rat hippocampi were fractionated by sucrose gradient ultracentrifugation. Individual fractions (lanes 1–9) were subjected to SDS-PAGE and Western blot analyses using anti-PABP (C) or anti-MKRN1 antibodies (D). The positions of molecular size marker proteins (in kDa) are indicated on the right. E, RNAs purified from individual gradient fractions were separated by agarose gel electrophoresis and stained with ethidium bromide. 28 S and 18 S, large and small ribosomal RNAs.
FIGURE 8.
FIGURE 8.
MKRN1-short is associated with dendritically localized mRNAs. A, shown are protein lysates from rat primary cortical neurons expressing recombinant EGFP-MKRN1-short, EGFP-PABP, or EGFP alone that were subjected to immunoprecipitation with GFP Trap®_A beads followed by SDS-PAGE and Western blot analyses using anti-GFP antibodies for detection of EGFP-MKRN1 (lanes 1 and 2), EGFP-PABP (lanes 3 and 4), and EGFP (lanes 5 and 6), respectively. I, input proteins (lanes 1, 3, and 5); E, immunoprecipitated proteins eluted from the beads (lanes 2, 4, and 6). The positions of molecular size marker proteins (in kDa) are indicated on the right. B, RNAs extracted from whole cell lysates (input) and immunoprecipitated protein fractions (eluate) were subjected to semiquantitative real-time RT-PCR using primers for MAP2-, Arc/Arg3.1-, and neuron-specific β-tubulin 3 cDNAs. The graph depicts the enrichment of individual transcripts in inputs and in eluates obtained by immunoprecipitation of EGFP-MKRN1-short (open bars) and EGFP-PABP (closed bars), respectively, compared with the empty vector control (EGFP-IP). Data were obtained using REST (relative expression software tool) 2008 software for group-wise comparison and statistical analysis of relative expression results in real-time PCR (41). S.E., vertical lines, enrichment/eluates, p < 0.001; enrichment/inputs, not significant. Arc/Arg3.1, activity-regulated cytoskeleton-associated.
FIGURE 9.
FIGURE 9.
Induction of LTP in the perforant pathway leads to accumulation of MKRN1-short in the stimulated dendritic lamina of the dentate gyrus. Brain sections from rats subjected to plasticity-inducing unilateral stimulation of the perforant pathway were immunolabeled with anti-MKRN1-short antibodies (upper panel) and anti-PABP antibodies (lower panel), respectively. Arrowheads point to MKRN1-short accumulation in the middle molecular layer (MML) of the dentate gyrus. GCL, granule cell layer; IML, inner molecular layer; OML, outer molecular layer.
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