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The nifk gene is widely expressed in mouse tissues and is up-regulated in denervated hind limb muscle
Cell Biology International
Cell Biology International 27 (2003) 469–475
www.elsevier.com/locate/cellbi
The nifk gene is widely expressed in mouse tissues and is up-regulated in denervated hind limb muscle Caroline Magnusson 1*, Marlene Norrby 1, Rolf Libelius 2, Sven Ta˚gerud 1 1
2
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden Department of Pharmacology and Clinical Neuroscience, Division of Clinical Neurophysiology, Umea˚ University Hospital, SE-901 85 Umea˚, Sweden Received 14 October 2002; revised 20 December 2002; accepted 12 February 2003
Abstract Denervation of skeletal muscle alters the expression of many genes, which may be important for establishing optimal conditions for reinnervation. Using the differential display technique we have attempted to discover neurally regulated genes in skeletal muscle. An mRNA that is up-regulated in denervated hind limb muscle was identified and cloned. The cDNA encodes an RNA-binding protein, which was discovered during the course of this work to be a nucleolar protein interacting with the fork-head associated domain of the proliferation marker protein Ki-67, and named NIFK. We show that the nifk gene is widely expressed in adult mouse tissues and that the expression is up-regulated in denervated hind limb muscle. No difference between expression in perisynaptic and extrasynaptic portions of muscle was observed. The widespread expression in adult tissues suggests that the NIFK protein has other functions in addition to its interaction with Ki-67, which is only expressed in proliferating cells. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Gene expression; Differential display; Denervation; Hemidiaphragm; RNA binding protein and screening
1. Introduction Denervation of skeletal muscle alters the expression of many genes. Thus, denervation leads to the reappearance of the fetal acetylcholine receptor subunit gamma (AChR) in muscle (Witzemann et al., 1987) and an up-regulation of mRNAs encoding e.g. brain-derived neurotrophic factor (BDNF; Funakoshi et al., 1993; Koliatsos et al., 1993), glial cell line-derived neurotrophic factor (GDNF; Lie and Weis, 1998; Naveilhan et al., 1997) and neural cell adhesion molecule (N-CAM; Tews et al., 1997). Increased endocytosis occurs in the perisynaptic region of denervated muscle (Elmquist et al., 1992; Libelius and Ta˚gerud, 1984) that may reflect exocytotic activity resulting from secretion of neurotrophic factor(s) (Vult von Steyern et al., 1993). A proposed function of these factors is the guiding of regenerating nerves to the original synapse region, which * Corresponding author. Tel.: +46-480-44-62-39; fax: +46-480-44-62-62. E-mail address: [email protected] (C. Magnusson).
is the preferred site for reinnervation (Bennett and Pettigrew, 1976; Taxt, 1983). We have used the differential display technique (Liang and Pardee, 1992) in an attempt to discover neurally regulated genes in skeletal muscle. An mRNA with differential expression in innervated and denervated muscle was discovered and the full length cDNA was cloned by PCR. The cDNA encodes an RNA-binding protein which was independently discovered during the course of this work and named NIFK: nucleolar protein interacting with the fork-head associated (FHA) domain of protein Ki-67 (Takagi et al., 2001). Human NIFK (hNIFK) was discovered in a two-hybrid screening from a HeLa cDNA library, using the FHA domain of protein Ki-67, a cell proliferation marker of unknown function, as bait. NIFK was thought to interact with protein Ki-67 in a mitosis-specific and phosphorylationdependent manner (Takagi et al., 2001). The corresponding mouse cDNA (mNIFK) was cloned by PCR using primers deduced from the expressed sequence tags (EST) database. The mouse sequence was similar, but not identical, to a sequence published by the RIKEN
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00038-6
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group, resulting in differences in the predicted amino acid sequence of the carboxy-terminal part of the protein (see Takagi et al., 2001). In the present study we confirm the mouse NIFK protein sequence suggested by Takagi et al. (2001), and also provide the complete 3#-untranslated region of the cDNA. We further show that the nifk gene is widely expressed in mouse tissues and that the expression in hind limb skeletal muscle is up-regulated following denervation.
MgCl2 and 10 mM dithiothreitol (DTT). The mixture was incubated at 65 (C for 5 min followed by 37 (C for 10 min. 100 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Promega, Madison, WI, USA) were then added and the incubation was continued for an additional 50 min at 37 (C followed by 75 (C for 5 min. As controls for contaminating DNA, separate tubes containing all components except the reverse transcriptase were prepared. 2.3. PCR amplification of cDNA
2. Materials and Methods 2.1. Denervation Adult male NMRI mice (about 30 g) were used in all experiments. Denervation of right hind limb muscles was performed on anaesthetized (with a mixture of ketamine and xylazine, i.p.) animals by sectioning the sciatic nerve at the mid-thigh level. Before removing a few mm of the nerve, a drop of xylocain (20 mg/ml) was applied to the nerve and left for a few minutes, thus reducing neural activity when sectioning it. Lower hind limb muscles (gastrocnemius, soleus, anterior tibial, and extensor digitorum longus) were dissected and pooled 1–10 days after denervation. Innervated contralateral muscles were used as controls. Denervation of the left hemidiaphragm was performed through a small incision penetrating the skin and the intercostal muscles between the lower ribs. The phrenic nerve was cleared from below the heart with a glass hook, pulled out through the incision and sectioned. Six days after denervation the denervated hemidiaphragm was dissected and collected in phosphate buffered saline (PBS) with calcium (2 mM). Regions thought to contain the endplates (about the mid third of the muscle, the perisynaptic region) were dissected out under a microscope and pooled (6–8 muscles). Regions devoid of endplates (extrasynaptic regions) were pooled separately. Innervated left hemidiaphragms were used as controls. Perisynaptic and extrasynaptic regions were pooled from 6–20 muscles. 2.2. Differential display and reverse transcription of RNA The differential display technique used is based on Liang and Pardee (1992) original protocol and on protocols from Colonna-Romano et al. (1998). cDNA subpopulations were prepared from four independent reverse transcription reactions, each containing one of four oligo-dT12VN-primers (V is a mixture of A, C and G in equal amounts and N is one of A, C, G or T). For each reaction of 20 µl, 200 ng total RNA (see below) were mixed with 20 µM dNTPs, 0.2 µM primer (dT12VN, Genosys Biotechnologies Inc, Cambridge, UK), 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM
The PCR mixture (20 µl) contained 2 µl cDNA (see above), 2 µM dNTPs, 10 µCi [35S] dATP (10 mCi/ml, >1000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ, USA), 0.2 µM oligo-dT12VN-primer, 0.2 µM random decamer primer (5#-ACCTCAC TGG-3#, Pharmacia Biotech, Uppsala, Sweden), 1.25 µM MgCl2, 10 mM Tris–HCl (pH 8.3), 50 mM KCl and 0.05 U Taq polymerase (Perkin Elmer, Roche Molecular Systems Inc., Branchbury, NJ, USA). The mixture was prepared without enzyme and was overlaid with 50 µl mineral oil. After a denaturing incubation of 5 min at 95 (C the enzyme was added and amplification was performed in a Perkin Elmer Cetus DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, CT, USA) for 40 cycles of 94 (C for 30 s, 42 (C for 2 min, and 72 (C for 30 s followed by a final elongation step at 72 (C for 5 min. 11.5 µl loading solution containing 95% (v/v) formamide, 9 mg/ml bromophenol blue, 9 mg/ml Xylene cyanol FF and 10 mM EDTA were added to the PCR mix, and the cDNA was denatured at 80 (C for 5 min before 7 µl was loaded on to a gel (6% acrylamide:bisacrylamide (19:1), 7 M Urea, 90 mM Tris–Borate, and 2 mM EDTA). The gel was prerun at 1500 V for >30 min before sample loading and run for an additional 5–6 h in a 90 mM Tris–Borate solution with 2 mM EDTA. Gels were transferred on to filter paper (3 MM Chr, Whatman International Ltd, Maidstone, UK), dried under vacuum conditions and exposed to X-ray film for 1–8 days. Regions of interest were cut out from the gel and a new X-ray film was exposed to verify that the correct part of the gel had been excised. Gel slices were rehydrated in 100 µl dH2O for about 45 min at room temperature before boiling for 15 min. The cDNA extracted from the gel slice was precipitated with 450 µl ice-cold 95% ethanol, 10 µl 3 M sodium acetate (pH 5.2), and 5 µl 10 mg/ml glycogen (Boehringer Mannheim, Germany) as a carrier, and the pellet was redissolved in 10 µl dH2O. Reamplification was carried out on 4 µl of the eluted cDNA in a total volume of 40 µl, using the same PCR conditions as for the original amplification, except that 20 µM dNTPs were used and no radioactive nucleotides were included.
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2.4. Cloning and sequencing of cDNA fragment The reamplified cDNA fragment was cloned into the pGEM-T vector (Promega, Madison, WI, USA) and plasmids purified using Wizard Plus mini- and midiprep kits (Promega, Madison, WI, USA). Inserts were sequenced using an ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, CA, USA) and the Big Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (PE Applied Biosystems, Warrington, UK). Sequences were submitted to the National Center for Biotechnology Information (NCBI) for alignment and identification using the BLAST program (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi). 2.5. Northern blot analysis Muscle homogenization, RNA extraction, separation and hybridization were performed as described in Magnusson et al. (2001). 2.6. Transcription of
32
P-labeled RNA probes
Plasmids were linearized with Not I restriction enzyme and an antisense RNA probe was transcribed in 10 µl from 0.5 µg plasmid with 9.5 U T7 RNA polymerase (Promega, Madison, WI, USA) in 40 mM Tris–HCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 100 µg/ml BSA, 10 mM dithiothreitol (DTT), 10 U RNase inhibitor (Promega, Madison, WI, USA), 0.5 mM rA/G/C/UTP, and 30 µCi of either [32P]UTP or [32P]CTP (20 mCi/ml, >800 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ, USA). The labeled nucleotide replaced the unlabeled nucleotide in the reaction. Incorporation efficiency was estimated after incubation for 1 h at 37 (C by applying 0.5 µl of the reaction mix on each of two Whatman DE81 filters, one of which was washed for 20 min at room temperature in 5% Na2HPO4. The filters were analyzed in a Beckman LS 6000 SE scintillation counter to measure incorporated radioactivity and incorporation efficiency. A cDNA fragment of the AChR subunit was cloned as previously described (Magnusson et al., 2001) using primers (forward primer: 5#-CGG GAT CCA GCT GTT ACG GAT GCA TG, bold letters are restriction site for BamHI, reverse primer: 5#-GCC CAA GCT TGC TTC AGG CTG CCA CAG A, bold letters are restriction site for Hind III) deduced from the GenBank sequence with accession number M30514. A probe (252 nucleotides) was transcribed as described above after linearization with BamHI. The probe sequence corresponds to nucleotides 1111–1362 in Boulter et al. (1986). 2.7. Cloning and sequencing of the entire cDNA 3# and 5# RACE reactions were performed with Gibco BRL kits (Life Technologies Inc., Gaithersburg,
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MD, USA) according to the manufacturer’s protocol. Gene specific primers were designed from sequences obtained in the differential display experiment and purchased from Eurogentec, Seraing, Belgium. A 5# specific primer was designed from EST-database sequences and used for PCR amplification of the 5# terminus of the mRNA. The whole cDNA of 1645 nucleotides, in which each nucleotide was sequenced at least four times, has been submitted to GenBank with accession number AY030275. 2.8. Data analysis and statistics For quantitation of gene expression from Northern blots, autoradiograms were illuminated on a Kaiser prolite 5000 lightbox (Kaiser Fototechnik GmbH & Co. KG, Buchen, Germany) and recorded using a Sony XC-75CE CCD Video camera module (Sony Corporation, Kanagawa-ken, Japan) with a Cosmicar/ Pentax TV zoom lens 8–48 mm (Pentax Precision Co. Ltd, Tokyo, Japan). Images were captured using the Techtum ImageSaver 32 program (Techtum Lab. AB, Umea˚, Sweden). Image analysis was performed on a Macintosh computer using the gel plotting macro of the public domain NIH Image program (1.62, developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Results were obtained in uncalibrated optical density units. For quantitation of gene expression in hind limb muscle, autoradiograms from five separate Northern blots were analyzed. Each of these blots contained at least one innervated muscle extract, and the innervated signal was used as a reference in the autoradiogram. The gene expression in denervated muscle was then calculated relative to the innervated reference signal. If Northern blots contained two or more lanes with extracts from innervated muscles, the average signal from innervated muscles was used as the reference signal. Similarly, if Northern blots contained two or more lanes of denervated muscle extracts (same day after denervation), the average signal was used. The total number of extracts from different muscles included in the analysis were: seven innervated, two 1-day denervated, five 3-day denervated, four 6-day denervated and four 10-day denervated. A similar method was used to determine gene expression in denervated hemidiaphragm muscle relative to that in innervated muscle. Data are presented as mean valuesstandard error of the mean (SEM). Analysis of variance (ANOVA) was used to test for statistically significant differences between expression in muscles at different times after denervation. Since no statistically significant difference was observed, the expression data from all denervated muscles were pooled and Student’s (one sample) t-test
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Fig. 1. NIFK mRNA expression in innervated (Inn) and 6-day denervated (Den) hind limb muscles, liver (L), skin (Sk), brain (B), heart (H), intestine (I), and spleen (Sp). The figure shows autoradiogram (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 20 µg. The autoradiogram was exposed for 11 days at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
Fig. 2. NIFK mRNA expression in innervated (Inn) and 3–10-day (3 d–10 d) denervated (Den) hind limb muscles. The figure shows autoradiogram (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 15 µg. The autoradiogram was exposed for 4 days at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
was used to determine whether the mean expression in denervated muscle was significantly different from 1.00 (the normalized expression in innervated muscle).
3. Results A cDNA fragment, which appeared to be differentially expressed in innervated and denervated muscle was isolated from a differential display gel, cloned into a pGEM-T vector and sequenced. The fragment was 319 nucleotides long (including primers) and contained no oligo-dT12VN-primer as a reverse primer, but instead had the random decamer primer at both ends. 3.1. Northern blots Northern blot hybridizations revealed a single band slightly smaller than the 18S rRNA (1869 nucleotides, Figs. 1, 2 and 4A). The mRNA was expressed in all tissues examined, as shown in Fig. 1. In hind limb muscle the expression was up-regulated after denervation, as shown in Figs. 1 and 2. Quantitative analysis of the mRNA expression in hind limb muscle at different times after denervation is shown in Fig. 3, in which expression in denervated muscle is presented relative to that in innervated muscle. Analysis of variance (ANOVA) did not detect any statistically significant differences between the expression 1 day (1.540.43, n=2), 3 days (2.800.33, n=4), 6 days (3.680.17, n=3) or 10 days (3.030.68, n=3) after denervation. The
Fig. 3. NIFK mRNA expression in 1–10-day denervated hind limb muscles. Expression levels are depicted relative to the expression in innervated muscles (dashed line). Numbers in brackets indicate the number of observations. See text for statistical analysis.
expression values for all denervated muscles were therefore pooled and the mean expression calculated as 2.870.28, n=12. This expression is significantly (P<0.001) higher than 1.00, the normalized expression in innervated muscle. In order to examine whether or not the gene was differentially expressed in different parts of muscle, the probe was hybridized to membranes with RNA from separated perisynaptic (endplate rich) and extrasynaptic (devoid of endplates) parts of innervated and denervated hemidiaphragms (Fig. 4A). No difference was observed
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Fig. 4. Expression of NIFK mRNA (A) and AChR subunit mRNA (B) in extrasynaptic (ES) and perisynaptic (PS) regions of innervated (Inn) and 6-day denervated (Den) hemidiaphragm muscle. The figure shows autoradiograms (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 15 µg. The same RNA preparation was used in A and B. The autoradiograms were exposed for 3 days (A) and 5 days (B) at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
in expression between perisynaptic and extrasynaptic regions of the muscle. The expression in denervated relative to innervated hemidiaphragm was not significantly increased 6 days after denervation (1.240.17, n=3). For comparison and confirmation of successful denervation of hemidiaphragms, Fig. 4B shows the expression of AChR mRNA in the same RNA preparation as were used for Fig. 4A. The AChR subunit is not expressed in innervated muscle but is strongly expressed following denervation. 3.2. Cloning, sequencing and translation of the cDNA A cDNA of 1645 nucleotides was cloned using the RACE technique and PCR amplification. The cDNA was sequenced (Fig. 5) and submitted to GenBank (accession number AY030275). The 5# terminus (about 95 nt) could not be obtained with the RACE method but was PCR amplified using a 5#-primer (20 nt) designed from EST-database sequences (Fig. 5). Starting from an initiator methionine within a good Kozak consensus sequence (Kozak, 1986), an open reading frame was identified from nucleotide number 22 to 975 (Fig. 5). A possible polyadenylation signal [AAUAUA] (Beaudoing et al., 2000; Graber et al., 1999) was present 22 nucleotides upstream of the poly-A tail (Fig. 5). During the course of this work two sequences similar to the full-length cDNA were submitted to GenBank
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with the accession numbers AK009440 (RIKEN group) and AB056870 (Takagi et al., 2001), both cloned from mouse material. In Fig. 5 the nucleotide sequence obtained in the present study and the translated amino acid sequence are shown. Two nucleotides differ between this sequence and the one reported by Takagi et al. (2001), but in both cases the same amino acid results in the translated sequence. The first divergence occurs at nucleotide 27, where a cytosine (C) in the present sequence is replaced by a thymine (T) in the previous NIFK sequence. Both combinations encode alanine. The second variant is at nucleotide 759; our sequence displays a guanine (G) while an adenine (A) is present in the previous NIFK sequence. Both combinations will encode arginine. In the sequence reported by the RIKEN group, there is an insertion of two nucleotides that disrupt the amino acid sequence 12 amino acids from the C-terminal. This insertion was not observed by Takagi et al. (2001) and is not present in the sequence obtained in this study.
4. Discussion A cDNA fragment obtained using the differential display technique was cloned and the full-length cDNA analyzed. The sequence was found to be identical to that for NIFK, a recently discovered RNA-binding protein (Takagi et al., 2001). Northern blot analysis revealed expression of an mRNA slightly smaller than the 18S rRNA (1869 nucleotides in the mouse) in all tissues examined. This agrees well with the size of the cloned cDNA, which was about 1630 nucleotides excluding the poly-A tail. Northern blot hybridizations detected a higher mRNA expression in denervated hind limb muscle than in innervated muscle. No difference was observed between perisynaptic and extrasynaptic portions of hemidiaphragm muscle, suggesting that the mRNA is transcribed throughout the muscle and not localized to areas of neuromuscular contact. No statistically significant increase in expression was observed after denervation of hemidiaphragm muscle. The reason for this may be related to the differences between hemidiaphragm and hind limb muscles in their responses to denervation. While hind limb muscles become atrophic and inactive after denervation, the hemidiaphragm becomes transiently hypertrophic, possibly as a result of passive stretching (Feng and Lu, 1965; Gutmann et al., 1966; Sola and Martin, 1953; Zhan and Sieck, 1992; Zhan et al., 1995). In both hemidiaphragm and hind limb muscles, an increase in RNA content occurs after denervation (Bowman and Martin, 1971; Goldspink, 1976; Goldspink et al., 1983; Little et al., 1982; Manchester and Harris, 1968; Yang et al., 1998). However, this
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Fig. 5. Nucleotide sequence of the cloned cDNA and translated amino acid sequence. The underlined sequence (dashed line) is the primer sequence used to obtain the 5#-end of the cDNA. The start and stop codons are marked in bold and the riboprobe-sequence used for Northern blotting is in italics and bold. A possible polyadenylation signal [AAUAUA] is present 22 nucleotides upstream of the poly-A start and is marked by a letter-midline. The translated open reading frame is shown below the nucleotide sequence, 317 amino acids correspond to nucleotides 22–972 in the cDNA. The motif resembling an RNA binding domain (RBD) is underlined in the nucleotide sequence.
increase is much larger in hemidiaphragm muscle than in hind limb muscle. In our experience, 6-day denervated hind limb muscles contain about 1.6 times the total RNA content of contralateral innervated muscles, whereas 6-day denervated left hemidiaphragms contain about three times the total RNA content of innervated controls (see Magnusson et al., 2001). In the present study the amount of RNA loaded on gels was normalized to equal amounts of total RNA (mainly ribosomal RNA). Thus, only genes in the hemidiaphragm that show an increase in total muscle expression of more than about three times will be detected as being up-regulated after denervation. Two nucleotides were found to differ between the cDNA of the present study and the recently reported nifk sequence (Takagi et al., 2001) with GenBank accession number AB056870. However, they do not result in any differences in the translated protein sequence and may well be due to strain variations in the animals or cells used. The present study thus confirms the amino acid sequence reported by Takagi et al. (2001) and furthermore provides the entire 3#-untranslated region of the cDNA. In this region the less common
polyadenylation signal [AAUAUA] was found instead of the more common [AAUAAA] signal. The former has been reported in both humans and mice (Graber et al., 1999). The cDNA identified in the present study thus encodes a protein identical to the recently described NIFK, which is an RNA binding protein (Takagi et al., 2001). One or several RNA recognition motifs are found in a variety of RNA-binding proteins, including hnRNP particles, translation factors, snRNPs, proteins involved in pre-mRNA and pre-rRNA processing and poly(A)binding proteins (Birney et al., 1993). The human NIFK protein was discovered due to its ability to interact with Ki-67, a protein expressed only in proliferating cells (see Scholzen and Gerdes, 2000). This interaction of NIFK with Ki-67 appears to be dependent on phosphorylation of NIFK (Takagi et al., 2001). In the present study the mouse nifk gene is shown to be expressed as a single transcript in all adult tissues examined. Expression in tissues such as brain and skeletal muscle that contain mainly non-proliferating cells suggests a more fundamental function of the NIFK protein in addition to its potential role in cell proliferation.
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Acknowledgements We are most grateful to Dr Marc Van Ranst, Katholieke Universiteit Leuven, Belgium, for assistance with bioinformatics and feedback on the manuscript. Janna Holmgren and Nina Lindblom are appreciated for help with cloning the AChR cDNA. This work was supported by grants from the Natural Sciences Faculty, University of Kalmar, Sweden and by a grant from Umea˚ University Hospital, Clinical Neuroscience Research Fund, Sweden.
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Cell Biology International 27 (2003) 469–475
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The nifk gene is widely expressed in mouse tissues and is up-regulated in denervated hind limb muscle Caroline Magnusson 1*, Marlene Norrby 1, Rolf Libelius 2, Sven Ta˚gerud 1 1
2
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden Department of Pharmacology and Clinical Neuroscience, Division of Clinical Neurophysiology, Umea˚ University Hospital, SE-901 85 Umea˚, Sweden Received 14 October 2002; revised 20 December 2002; accepted 12 February 2003
Abstract Denervation of skeletal muscle alters the expression of many genes, which may be important for establishing optimal conditions for reinnervation. Using the differential display technique we have attempted to discover neurally regulated genes in skeletal muscle. An mRNA that is up-regulated in denervated hind limb muscle was identified and cloned. The cDNA encodes an RNA-binding protein, which was discovered during the course of this work to be a nucleolar protein interacting with the fork-head associated domain of the proliferation marker protein Ki-67, and named NIFK. We show that the nifk gene is widely expressed in adult mouse tissues and that the expression is up-regulated in denervated hind limb muscle. No difference between expression in perisynaptic and extrasynaptic portions of muscle was observed. The widespread expression in adult tissues suggests that the NIFK protein has other functions in addition to its interaction with Ki-67, which is only expressed in proliferating cells. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Gene expression; Differential display; Denervation; Hemidiaphragm; RNA binding protein and screening
1. Introduction Denervation of skeletal muscle alters the expression of many genes. Thus, denervation leads to the reappearance of the fetal acetylcholine receptor subunit gamma (AChR) in muscle (Witzemann et al., 1987) and an up-regulation of mRNAs encoding e.g. brain-derived neurotrophic factor (BDNF; Funakoshi et al., 1993; Koliatsos et al., 1993), glial cell line-derived neurotrophic factor (GDNF; Lie and Weis, 1998; Naveilhan et al., 1997) and neural cell adhesion molecule (N-CAM; Tews et al., 1997). Increased endocytosis occurs in the perisynaptic region of denervated muscle (Elmquist et al., 1992; Libelius and Ta˚gerud, 1984) that may reflect exocytotic activity resulting from secretion of neurotrophic factor(s) (Vult von Steyern et al., 1993). A proposed function of these factors is the guiding of regenerating nerves to the original synapse region, which * Corresponding author. Tel.: +46-480-44-62-39; fax: +46-480-44-62-62. E-mail address: [email protected] (C. Magnusson).
is the preferred site for reinnervation (Bennett and Pettigrew, 1976; Taxt, 1983). We have used the differential display technique (Liang and Pardee, 1992) in an attempt to discover neurally regulated genes in skeletal muscle. An mRNA with differential expression in innervated and denervated muscle was discovered and the full length cDNA was cloned by PCR. The cDNA encodes an RNA-binding protein which was independently discovered during the course of this work and named NIFK: nucleolar protein interacting with the fork-head associated (FHA) domain of protein Ki-67 (Takagi et al., 2001). Human NIFK (hNIFK) was discovered in a two-hybrid screening from a HeLa cDNA library, using the FHA domain of protein Ki-67, a cell proliferation marker of unknown function, as bait. NIFK was thought to interact with protein Ki-67 in a mitosis-specific and phosphorylationdependent manner (Takagi et al., 2001). The corresponding mouse cDNA (mNIFK) was cloned by PCR using primers deduced from the expressed sequence tags (EST) database. The mouse sequence was similar, but not identical, to a sequence published by the RIKEN
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00038-6
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group, resulting in differences in the predicted amino acid sequence of the carboxy-terminal part of the protein (see Takagi et al., 2001). In the present study we confirm the mouse NIFK protein sequence suggested by Takagi et al. (2001), and also provide the complete 3#-untranslated region of the cDNA. We further show that the nifk gene is widely expressed in mouse tissues and that the expression in hind limb skeletal muscle is up-regulated following denervation.
MgCl2 and 10 mM dithiothreitol (DTT). The mixture was incubated at 65 (C for 5 min followed by 37 (C for 10 min. 100 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Promega, Madison, WI, USA) were then added and the incubation was continued for an additional 50 min at 37 (C followed by 75 (C for 5 min. As controls for contaminating DNA, separate tubes containing all components except the reverse transcriptase were prepared. 2.3. PCR amplification of cDNA
2. Materials and Methods 2.1. Denervation Adult male NMRI mice (about 30 g) were used in all experiments. Denervation of right hind limb muscles was performed on anaesthetized (with a mixture of ketamine and xylazine, i.p.) animals by sectioning the sciatic nerve at the mid-thigh level. Before removing a few mm of the nerve, a drop of xylocain (20 mg/ml) was applied to the nerve and left for a few minutes, thus reducing neural activity when sectioning it. Lower hind limb muscles (gastrocnemius, soleus, anterior tibial, and extensor digitorum longus) were dissected and pooled 1–10 days after denervation. Innervated contralateral muscles were used as controls. Denervation of the left hemidiaphragm was performed through a small incision penetrating the skin and the intercostal muscles between the lower ribs. The phrenic nerve was cleared from below the heart with a glass hook, pulled out through the incision and sectioned. Six days after denervation the denervated hemidiaphragm was dissected and collected in phosphate buffered saline (PBS) with calcium (2 mM). Regions thought to contain the endplates (about the mid third of the muscle, the perisynaptic region) were dissected out under a microscope and pooled (6–8 muscles). Regions devoid of endplates (extrasynaptic regions) were pooled separately. Innervated left hemidiaphragms were used as controls. Perisynaptic and extrasynaptic regions were pooled from 6–20 muscles. 2.2. Differential display and reverse transcription of RNA The differential display technique used is based on Liang and Pardee (1992) original protocol and on protocols from Colonna-Romano et al. (1998). cDNA subpopulations were prepared from four independent reverse transcription reactions, each containing one of four oligo-dT12VN-primers (V is a mixture of A, C and G in equal amounts and N is one of A, C, G or T). For each reaction of 20 µl, 200 ng total RNA (see below) were mixed with 20 µM dNTPs, 0.2 µM primer (dT12VN, Genosys Biotechnologies Inc, Cambridge, UK), 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM
The PCR mixture (20 µl) contained 2 µl cDNA (see above), 2 µM dNTPs, 10 µCi [35S] dATP (10 mCi/ml, >1000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ, USA), 0.2 µM oligo-dT12VN-primer, 0.2 µM random decamer primer (5#-ACCTCAC TGG-3#, Pharmacia Biotech, Uppsala, Sweden), 1.25 µM MgCl2, 10 mM Tris–HCl (pH 8.3), 50 mM KCl and 0.05 U Taq polymerase (Perkin Elmer, Roche Molecular Systems Inc., Branchbury, NJ, USA). The mixture was prepared without enzyme and was overlaid with 50 µl mineral oil. After a denaturing incubation of 5 min at 95 (C the enzyme was added and amplification was performed in a Perkin Elmer Cetus DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, CT, USA) for 40 cycles of 94 (C for 30 s, 42 (C for 2 min, and 72 (C for 30 s followed by a final elongation step at 72 (C for 5 min. 11.5 µl loading solution containing 95% (v/v) formamide, 9 mg/ml bromophenol blue, 9 mg/ml Xylene cyanol FF and 10 mM EDTA were added to the PCR mix, and the cDNA was denatured at 80 (C for 5 min before 7 µl was loaded on to a gel (6% acrylamide:bisacrylamide (19:1), 7 M Urea, 90 mM Tris–Borate, and 2 mM EDTA). The gel was prerun at 1500 V for >30 min before sample loading and run for an additional 5–6 h in a 90 mM Tris–Borate solution with 2 mM EDTA. Gels were transferred on to filter paper (3 MM Chr, Whatman International Ltd, Maidstone, UK), dried under vacuum conditions and exposed to X-ray film for 1–8 days. Regions of interest were cut out from the gel and a new X-ray film was exposed to verify that the correct part of the gel had been excised. Gel slices were rehydrated in 100 µl dH2O for about 45 min at room temperature before boiling for 15 min. The cDNA extracted from the gel slice was precipitated with 450 µl ice-cold 95% ethanol, 10 µl 3 M sodium acetate (pH 5.2), and 5 µl 10 mg/ml glycogen (Boehringer Mannheim, Germany) as a carrier, and the pellet was redissolved in 10 µl dH2O. Reamplification was carried out on 4 µl of the eluted cDNA in a total volume of 40 µl, using the same PCR conditions as for the original amplification, except that 20 µM dNTPs were used and no radioactive nucleotides were included.
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2.4. Cloning and sequencing of cDNA fragment The reamplified cDNA fragment was cloned into the pGEM-T vector (Promega, Madison, WI, USA) and plasmids purified using Wizard Plus mini- and midiprep kits (Promega, Madison, WI, USA). Inserts were sequenced using an ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, CA, USA) and the Big Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (PE Applied Biosystems, Warrington, UK). Sequences were submitted to the National Center for Biotechnology Information (NCBI) for alignment and identification using the BLAST program (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi). 2.5. Northern blot analysis Muscle homogenization, RNA extraction, separation and hybridization were performed as described in Magnusson et al. (2001). 2.6. Transcription of
32
P-labeled RNA probes
Plasmids were linearized with Not I restriction enzyme and an antisense RNA probe was transcribed in 10 µl from 0.5 µg plasmid with 9.5 U T7 RNA polymerase (Promega, Madison, WI, USA) in 40 mM Tris–HCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 100 µg/ml BSA, 10 mM dithiothreitol (DTT), 10 U RNase inhibitor (Promega, Madison, WI, USA), 0.5 mM rA/G/C/UTP, and 30 µCi of either [32P]UTP or [32P]CTP (20 mCi/ml, >800 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ, USA). The labeled nucleotide replaced the unlabeled nucleotide in the reaction. Incorporation efficiency was estimated after incubation for 1 h at 37 (C by applying 0.5 µl of the reaction mix on each of two Whatman DE81 filters, one of which was washed for 20 min at room temperature in 5% Na2HPO4. The filters were analyzed in a Beckman LS 6000 SE scintillation counter to measure incorporated radioactivity and incorporation efficiency. A cDNA fragment of the AChR subunit was cloned as previously described (Magnusson et al., 2001) using primers (forward primer: 5#-CGG GAT CCA GCT GTT ACG GAT GCA TG, bold letters are restriction site for BamHI, reverse primer: 5#-GCC CAA GCT TGC TTC AGG CTG CCA CAG A, bold letters are restriction site for Hind III) deduced from the GenBank sequence with accession number M30514. A probe (252 nucleotides) was transcribed as described above after linearization with BamHI. The probe sequence corresponds to nucleotides 1111–1362 in Boulter et al. (1986). 2.7. Cloning and sequencing of the entire cDNA 3# and 5# RACE reactions were performed with Gibco BRL kits (Life Technologies Inc., Gaithersburg,
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MD, USA) according to the manufacturer’s protocol. Gene specific primers were designed from sequences obtained in the differential display experiment and purchased from Eurogentec, Seraing, Belgium. A 5# specific primer was designed from EST-database sequences and used for PCR amplification of the 5# terminus of the mRNA. The whole cDNA of 1645 nucleotides, in which each nucleotide was sequenced at least four times, has been submitted to GenBank with accession number AY030275. 2.8. Data analysis and statistics For quantitation of gene expression from Northern blots, autoradiograms were illuminated on a Kaiser prolite 5000 lightbox (Kaiser Fototechnik GmbH & Co. KG, Buchen, Germany) and recorded using a Sony XC-75CE CCD Video camera module (Sony Corporation, Kanagawa-ken, Japan) with a Cosmicar/ Pentax TV zoom lens 8–48 mm (Pentax Precision Co. Ltd, Tokyo, Japan). Images were captured using the Techtum ImageSaver 32 program (Techtum Lab. AB, Umea˚, Sweden). Image analysis was performed on a Macintosh computer using the gel plotting macro of the public domain NIH Image program (1.62, developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Results were obtained in uncalibrated optical density units. For quantitation of gene expression in hind limb muscle, autoradiograms from five separate Northern blots were analyzed. Each of these blots contained at least one innervated muscle extract, and the innervated signal was used as a reference in the autoradiogram. The gene expression in denervated muscle was then calculated relative to the innervated reference signal. If Northern blots contained two or more lanes with extracts from innervated muscles, the average signal from innervated muscles was used as the reference signal. Similarly, if Northern blots contained two or more lanes of denervated muscle extracts (same day after denervation), the average signal was used. The total number of extracts from different muscles included in the analysis were: seven innervated, two 1-day denervated, five 3-day denervated, four 6-day denervated and four 10-day denervated. A similar method was used to determine gene expression in denervated hemidiaphragm muscle relative to that in innervated muscle. Data are presented as mean valuesstandard error of the mean (SEM). Analysis of variance (ANOVA) was used to test for statistically significant differences between expression in muscles at different times after denervation. Since no statistically significant difference was observed, the expression data from all denervated muscles were pooled and Student’s (one sample) t-test
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Fig. 1. NIFK mRNA expression in innervated (Inn) and 6-day denervated (Den) hind limb muscles, liver (L), skin (Sk), brain (B), heart (H), intestine (I), and spleen (Sp). The figure shows autoradiogram (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 20 µg. The autoradiogram was exposed for 11 days at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
Fig. 2. NIFK mRNA expression in innervated (Inn) and 3–10-day (3 d–10 d) denervated (Den) hind limb muscles. The figure shows autoradiogram (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 15 µg. The autoradiogram was exposed for 4 days at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
was used to determine whether the mean expression in denervated muscle was significantly different from 1.00 (the normalized expression in innervated muscle).
3. Results A cDNA fragment, which appeared to be differentially expressed in innervated and denervated muscle was isolated from a differential display gel, cloned into a pGEM-T vector and sequenced. The fragment was 319 nucleotides long (including primers) and contained no oligo-dT12VN-primer as a reverse primer, but instead had the random decamer primer at both ends. 3.1. Northern blots Northern blot hybridizations revealed a single band slightly smaller than the 18S rRNA (1869 nucleotides, Figs. 1, 2 and 4A). The mRNA was expressed in all tissues examined, as shown in Fig. 1. In hind limb muscle the expression was up-regulated after denervation, as shown in Figs. 1 and 2. Quantitative analysis of the mRNA expression in hind limb muscle at different times after denervation is shown in Fig. 3, in which expression in denervated muscle is presented relative to that in innervated muscle. Analysis of variance (ANOVA) did not detect any statistically significant differences between the expression 1 day (1.540.43, n=2), 3 days (2.800.33, n=4), 6 days (3.680.17, n=3) or 10 days (3.030.68, n=3) after denervation. The
Fig. 3. NIFK mRNA expression in 1–10-day denervated hind limb muscles. Expression levels are depicted relative to the expression in innervated muscles (dashed line). Numbers in brackets indicate the number of observations. See text for statistical analysis.
expression values for all denervated muscles were therefore pooled and the mean expression calculated as 2.870.28, n=12. This expression is significantly (P<0.001) higher than 1.00, the normalized expression in innervated muscle. In order to examine whether or not the gene was differentially expressed in different parts of muscle, the probe was hybridized to membranes with RNA from separated perisynaptic (endplate rich) and extrasynaptic (devoid of endplates) parts of innervated and denervated hemidiaphragms (Fig. 4A). No difference was observed
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Fig. 4. Expression of NIFK mRNA (A) and AChR subunit mRNA (B) in extrasynaptic (ES) and perisynaptic (PS) regions of innervated (Inn) and 6-day denervated (Den) hemidiaphragm muscle. The figure shows autoradiograms (top) and ethidium bromide fluorescence (bottom) for verification of RNA quality and loading normalization. The amount of total RNA loaded per lane was 15 µg. The same RNA preparation was used in A and B. The autoradiograms were exposed for 3 days (A) and 5 days (B) at 80 (C with intensifying screens. Positions of 28S and 18S ribosomal RNA are indicated with closed and open arrowheads, respectively.
in expression between perisynaptic and extrasynaptic regions of the muscle. The expression in denervated relative to innervated hemidiaphragm was not significantly increased 6 days after denervation (1.240.17, n=3). For comparison and confirmation of successful denervation of hemidiaphragms, Fig. 4B shows the expression of AChR mRNA in the same RNA preparation as were used for Fig. 4A. The AChR subunit is not expressed in innervated muscle but is strongly expressed following denervation. 3.2. Cloning, sequencing and translation of the cDNA A cDNA of 1645 nucleotides was cloned using the RACE technique and PCR amplification. The cDNA was sequenced (Fig. 5) and submitted to GenBank (accession number AY030275). The 5# terminus (about 95 nt) could not be obtained with the RACE method but was PCR amplified using a 5#-primer (20 nt) designed from EST-database sequences (Fig. 5). Starting from an initiator methionine within a good Kozak consensus sequence (Kozak, 1986), an open reading frame was identified from nucleotide number 22 to 975 (Fig. 5). A possible polyadenylation signal [AAUAUA] (Beaudoing et al., 2000; Graber et al., 1999) was present 22 nucleotides upstream of the poly-A tail (Fig. 5). During the course of this work two sequences similar to the full-length cDNA were submitted to GenBank
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with the accession numbers AK009440 (RIKEN group) and AB056870 (Takagi et al., 2001), both cloned from mouse material. In Fig. 5 the nucleotide sequence obtained in the present study and the translated amino acid sequence are shown. Two nucleotides differ between this sequence and the one reported by Takagi et al. (2001), but in both cases the same amino acid results in the translated sequence. The first divergence occurs at nucleotide 27, where a cytosine (C) in the present sequence is replaced by a thymine (T) in the previous NIFK sequence. Both combinations encode alanine. The second variant is at nucleotide 759; our sequence displays a guanine (G) while an adenine (A) is present in the previous NIFK sequence. Both combinations will encode arginine. In the sequence reported by the RIKEN group, there is an insertion of two nucleotides that disrupt the amino acid sequence 12 amino acids from the C-terminal. This insertion was not observed by Takagi et al. (2001) and is not present in the sequence obtained in this study.
4. Discussion A cDNA fragment obtained using the differential display technique was cloned and the full-length cDNA analyzed. The sequence was found to be identical to that for NIFK, a recently discovered RNA-binding protein (Takagi et al., 2001). Northern blot analysis revealed expression of an mRNA slightly smaller than the 18S rRNA (1869 nucleotides in the mouse) in all tissues examined. This agrees well with the size of the cloned cDNA, which was about 1630 nucleotides excluding the poly-A tail. Northern blot hybridizations detected a higher mRNA expression in denervated hind limb muscle than in innervated muscle. No difference was observed between perisynaptic and extrasynaptic portions of hemidiaphragm muscle, suggesting that the mRNA is transcribed throughout the muscle and not localized to areas of neuromuscular contact. No statistically significant increase in expression was observed after denervation of hemidiaphragm muscle. The reason for this may be related to the differences between hemidiaphragm and hind limb muscles in their responses to denervation. While hind limb muscles become atrophic and inactive after denervation, the hemidiaphragm becomes transiently hypertrophic, possibly as a result of passive stretching (Feng and Lu, 1965; Gutmann et al., 1966; Sola and Martin, 1953; Zhan and Sieck, 1992; Zhan et al., 1995). In both hemidiaphragm and hind limb muscles, an increase in RNA content occurs after denervation (Bowman and Martin, 1971; Goldspink, 1976; Goldspink et al., 1983; Little et al., 1982; Manchester and Harris, 1968; Yang et al., 1998). However, this
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Fig. 5. Nucleotide sequence of the cloned cDNA and translated amino acid sequence. The underlined sequence (dashed line) is the primer sequence used to obtain the 5#-end of the cDNA. The start and stop codons are marked in bold and the riboprobe-sequence used for Northern blotting is in italics and bold. A possible polyadenylation signal [AAUAUA] is present 22 nucleotides upstream of the poly-A start and is marked by a letter-midline. The translated open reading frame is shown below the nucleotide sequence, 317 amino acids correspond to nucleotides 22–972 in the cDNA. The motif resembling an RNA binding domain (RBD) is underlined in the nucleotide sequence.
increase is much larger in hemidiaphragm muscle than in hind limb muscle. In our experience, 6-day denervated hind limb muscles contain about 1.6 times the total RNA content of contralateral innervated muscles, whereas 6-day denervated left hemidiaphragms contain about three times the total RNA content of innervated controls (see Magnusson et al., 2001). In the present study the amount of RNA loaded on gels was normalized to equal amounts of total RNA (mainly ribosomal RNA). Thus, only genes in the hemidiaphragm that show an increase in total muscle expression of more than about three times will be detected as being up-regulated after denervation. Two nucleotides were found to differ between the cDNA of the present study and the recently reported nifk sequence (Takagi et al., 2001) with GenBank accession number AB056870. However, they do not result in any differences in the translated protein sequence and may well be due to strain variations in the animals or cells used. The present study thus confirms the amino acid sequence reported by Takagi et al. (2001) and furthermore provides the entire 3#-untranslated region of the cDNA. In this region the less common
polyadenylation signal [AAUAUA] was found instead of the more common [AAUAAA] signal. The former has been reported in both humans and mice (Graber et al., 1999). The cDNA identified in the present study thus encodes a protein identical to the recently described NIFK, which is an RNA binding protein (Takagi et al., 2001). One or several RNA recognition motifs are found in a variety of RNA-binding proteins, including hnRNP particles, translation factors, snRNPs, proteins involved in pre-mRNA and pre-rRNA processing and poly(A)binding proteins (Birney et al., 1993). The human NIFK protein was discovered due to its ability to interact with Ki-67, a protein expressed only in proliferating cells (see Scholzen and Gerdes, 2000). This interaction of NIFK with Ki-67 appears to be dependent on phosphorylation of NIFK (Takagi et al., 2001). In the present study the mouse nifk gene is shown to be expressed as a single transcript in all adult tissues examined. Expression in tissues such as brain and skeletal muscle that contain mainly non-proliferating cells suggests a more fundamental function of the NIFK protein in addition to its potential role in cell proliferation.
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Acknowledgements We are most grateful to Dr Marc Van Ranst, Katholieke Universiteit Leuven, Belgium, for assistance with bioinformatics and feedback on the manuscript. Janna Holmgren and Nina Lindblom are appreciated for help with cloning the AChR cDNA. This work was supported by grants from the Natural Sciences Faculty, University of Kalmar, Sweden and by a grant from Umea˚ University Hospital, Clinical Neuroscience Research Fund, Sweden.
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