Fig. 2. QKI stabilizes mRNA in a MBP 3′-UTR-dependent manner. (A) The MBP 3′-UTR conveys QKI-binding to the GFP reporter mRNA. In vitro RNA binding was performed using ³⁵S-labeled QKI and biotin-labeled mRNA as described in Materials and methods. Parallel triplicates of RNA-binding reactions were performed and the average binding of the triplicate was calculated in each experiment. Four independent experiments were carried out (n = 4). The binding by GFP–MBP mRNA was normalized to that by the GFP mRNA in each paired reaction, and the standard deviation is indicated on top of the column. ** indicates P < 0.01 using paired t-test. (B) Co-expression of QKI results in elevated expression of the GFP–MBP reporter mRNA. GFP and GFP–MBP cell lines with or without stable expression of Flag-QKI-6 (QK+ and QK– respectively) were generated. The top panel shows a representative RPA gel that illustrates the steady state level of the reporter mRNA and the housekeeping β-actin mRNA. The quantitative analysis of the reporter mRNA (normalized to β-actin) from five independent experiments (n = 5) is illustrated in the bottom panel. * indicates P < 0.05 by paired t-test. The inset shows immunoblot analysis of Flag-QKI-6 and GFP expression in these cells. (C) QKI expression attenuates the decay of the GFP–MBP reporter mRNA. Cells were treated with actinomycin D (10 µg/ml) and RPA was carried out to determine the level of the GFP–MBP reporter mRNA normalized to that of the 18S rRNA at indicated time points. The level of reporter mRNA at 0 h was set as 100% and the decay curves were derived from five independent experiments (n = 5). Standard error is indicated for each data point. * indicates P < 0.05 by standard t-test when comparing results derived from QK+ and QK– cells.

Fig. 4. QKI isoforms are phosphorylated by Src-PTKs. (A) Phos phorylation of QKI by Src and Fyn in vitro. Src-PTK-dependent [γ-³²P]ATP labeling of His-QKI isoforms and [³²P]Src due to autophosphorylation are indicated on the left. (B) Retarded migration of Src-treated [³⁵S]QKI-7 on SDS–PAGE (arrows on the right). The lower molecular weight [³⁵S]QKI-7 band is an N-terminal truncated product derived from internal initiated translation. (C) Tyrosine phosphorylation of Flag-QKI isoforms in HEK293T cells. SrcA (Src-Y527F), constitutively active kinase; SrcI (Src-K295R), inactive kinase; IP, immunoprecipitation; IB, immunoblot; Anti-PTyr, phosphotyrosine-specific antibody PY20.

Fig. 6. Identification of tyrosines that mediate Src-dependent regulation of QKI–RNA interaction. (A) The C-terminal tyrosine cluster mediates Src-dependent tyrosine phosphorylation of QKI (QKI–PTyr). Top panel, amino acids of QKI-7 (A281–V310) harboring the tyrosine cluster. Y285, 288, 290, 292 and 303 are marked Y_a, Y_b, Y_c, Y_d and Y_e for simplicity. Middle panel, Src-dependent [γ-³²P]ATP labeling of His-QKI-7. Phenylalanine substitutions at various Ys were obtained by site-directed mutagenesis and marked on top of each lane. Bottom panel, the quantity of His-QKI in each phosphorylation reaction was evaluated by immunoblot using anti-QKI antibody. (B) Mutation of the C-terminal tyrosine cluster blocks QKI–PTyr in HEK293T cells. The expression of Flag-QKI-7 and HA-tagged kinases in lysate (load), and Flag-QKI–PTyr in the immunoprecipitate (IP) was detected by immunoblot (IB). (C) Mutation of the C-terminal tyrosine cluster abrogates Src-dependent regulation of QKI–RNA interaction. RNA binding was performed as described in Figure 5. Standard error was indicated for each column; *, P < 0.05, paired t-test.

Fig. 8. Down-regulation of QKI–PTyr during the most active phase of myelinogenesis associates with MBP accumulation. (A) QKI–PTyr in the developing myelin and MBP expression during myelin development. His-QKI-7 was subjected to phosphorylation by Src-PTKs in brain stem myelin at indicated ages in the presence of [γ-³²P]ATP. The top panel shows an image of SDS–PAGE indicating the down-regulation of [³²P]QKI–Ptyr. The middle panel shows the concordant increase of MBP detected by immunoblot in isolated myelin during development. The multiple bands of MBP represent major MBP protein isoforms derived from alternative splicing of the MBP mRNA. The bottom panel shows β-actin in the re-probed immunoblot as a loading control. (B) Quantitative analysis of [³²P]QKI–PTyr in the developing myelin by PhosphorImager analysis (n = 4). The standard deviation was indicated. P < 0.001 by one way ANOVA; **, P < 0.01 when compared with QKI–PTyr in the isolated myelin derived from P7 brain stem.

Fig. 1. QKI is required for the rapid accumulation of MBP mRNA during accelerated myelinogenesis. (A) Schematic representation of QKI isoforms (QKI-5, -6 and -7), each with a distinct C-terminus due to alternative splicing of the qkI primary transcript. QUA1 and QUA2 together with the single KH domain form the RNA-binding domain of QKI; the proline-rich PXXP motifs and the tyrosine cluster at the C-terminus are illustrated. (B) Detection of QKI protein isoforms in the brain. The left panel indicates detection of all three QKI isoforms in the whole tissue lysate derived from wild-type P10 brain stem (BS). The right panel shows detection of QKI-6 and -7 in the post-nuclear extracts derived from brain stem and CAD1 cells. The blot was re-probed by the antibody against the housekeeping protein eIF5α as a loading control. (C) Diminished QKI expression during accelerated myelinogenesis results in delayed MBP mRNA accumulation. The top panel illustrates the MBP mRNA expression profile in qk^v/qk^v and wt/qk^v littermate controls. RPA was performed using total RNA derived from brain stems at P12, P20, P2M and P4M, and the MBP mRNA level was estimated by normalizing the PhosphorImager reading of MBP to that of GAPDH. The bottom panel shows the developmental profile of QKI protein expression in qk^v/qk^v and wt/qk^v littermate controls. Brain stem extracts were prepared at indicated ages and subjected to SDS–PAGE immunoblot using anti-QKI antibody. The housekeeping protein LDH was used as a loading control. The level of QKI in each lane was estimated by normalization of total QKI signals to the LDH signal using NIH imaging software and indicated at the bottom.

Fig. 3. Expression of QKI and MBP mRNA during active myelinogenesis. The QKI mRNA isoforms were detected by RPA using an antisense riboprobe corresponding to the 3′-coding region of QKI-7 as described in Materials and methods. The full-length probe was protected by QKI-7; the rest of the QKI mRNA isoforms, mainly QKI-5 and QKI-6, protect a shorter fragment of the riboprobe that was evaluated in the same RPA gel. The steady state level of QKI-7, -5 and -6, as well as that of the MBP mRNA in wild-type brain stem was evaluated by RPA at postnatal day 7, 12, 16, 20 and 28. The top panel shows a representative RPA gel for detecting QKI mRNA isoforms and the housekeeping GAPDH mRNA. The PhosphorImager reading of QKI mRNAs is normalized to that of GAPDH, plotted against age and superimposed with the RPA result of MBP mRNA obtained from the same animals in the bottom panel.

Fig. 5. Tyrosine phosphorylation of QKI by Src-PTKs negatively regulates the interactions between QKI and the MBP mRNA. (A) Src treatment attenuates the interactions between QKI-7 and the MBP mRNA. RNA binding and Src treatment of QKI-7 are described in Materials and methods. [³⁵S]QKI-7 input (ng) is plotted against RNA-bound [³⁵S]QKI-7 determined by scintillation counting (c.p.m.). (B) Src treatment negatively regulates the activity of all three QKI isofroms (Q5, Q6 and Q7) for binding MBP mRNA. Ten nanograms of ³⁵S-labeled protein estimated by comparison with known amounts of His-QKI on SDS–PAGE immunoblot was used in each reaction. For each isoform, the MBP mRNA-bound QKI from mock-treated reactions was defined as 100%, and MBP mRNA-bound QKI from Src-treated reactions was normalized to that of mock-treated parallel reaction. The lower panel shows a representative SDS–PAGE image of the RNA-bound QKI isoforms in Src- and mock-treated reactions. The captured QKI in each reaction accounts for ∼10% of the input after the stringent washes. (C) Src treatment specifically regulates the activity of QKI in binding MBP mRNA. Ten nanograms of ³⁵S-labeled QKI-7 or FMRP was used in each reaction. The number of experiments performed (n) is indicated. Standard error is indicated for each column; *, P < 0.05, paired t-test.

Fig. 7. The C-terminal tyrosine cluster is the target for Src-PTK-dependent tyrosine phosphorylation of QKI in the myelin compartment. (A) Detection of QKI–PTyr by immunoprecipitation (IP) from the post-nuclear extracts derived from P12 brain stem (BS) and CAD1. (+) and (–) indicate the presence or absence of the corresponding primary antibody in IP. (B) The C-terminal tyrosine cluster of QKI mediates QKI phosphorylation by kinases in isolated myelin. [γ-³²P]ATP labeling assay was carried out using wild-type or mutant His-QKI-7 in the presence of Src, isolated myelin (myl) or whole cytoplasmic extract (cyt) derived from normal brain stem as described in Materials and methods. (C) Tyrosine phosphorylation of QKI in the isolated myelin is mediated by the PPXP motif. The top panel shows amino acids in the C-terminus of QKI-7, containing a single PPXP motif (marked by a bold underline) and a few PXXP proline-rich motifs (underlined). In vitro phosphorylation was carried out in isolated myelin using wild-type His-QKI-7, mutant His-QKI-7 Y_a and the mutant His-QKI-7 DSH3 in which the prolines in the single PPXP motif were replaced by alanines. A representative SDS–PAGE illustrating ³²P-labeled QKI was shown on the left. The PhosphorImager reading of wild-type His-QKI-7 was defined as 100%, and the PhosphorImager reading of each mutant was normalized to that of wild type in parallel experiments. Statistic analysis was carried out for three independent experiments (n = 3) and shown on the right. *, P < 0.05; ***, P < 0.001 compared with wild-type His-QKI by standard t-test. (D) Inhibition of QKI–PTyr in the developing myelin by Src-PTK inhibitor. Concentrations for each inhibitor are indicated on top of the corresponding lanes.