Journal: Nature Communications

Article Title: QKI is a critical pre-mRNA alternative splicing regulator of cardiac myofibrillogenesis and contractile function

doi: 10.1038/s41467-020-20327-5

Figure Lengend Snippet: Inducible QKI5, QKI6, and QKI7 expression cells are established in H1-8 lines (H1-8- QKI5 ind , H1-8- QKI6 ind , H1-8- QKI7 ind ). Representative images of immunofluorescence staining of TNNT2 and ACTN2 in H1-8- QKI5 ind , H1-8- QKI6 ind , H1-8- QKI7 ind cardiomyocytes with or without doxycycline induction. Myofibrillar structures are recovered in doxycycline-induced H1-8- QKI5 ind cardiomyocytes (Day-15) and ACTN2 expression is also recovered. However, doxycycline-induced H1-8- QKI6 ind and H1-8- QKI7 ind cardiomyocytes fail to recover ACTN2 expression and generate normal myofibrillar structures (Day-15). Scale bar: 25 μm. All experiments are independently repeated three times with three different sets of cell samples to confirm the reproducibility of the findings.

Article Snippet: The membranes were blocked with 5% nonfat dry milk (Bio-Rad, 1706404) in Tris-buffered saline containing Tween-20 and incubated with primary antibodies against TNNT2 (DSHB, Cat#AB528495), ACTN2 (Sigma, Cat#A7811), MF20 (DSHB, Cat#AB2147781), MYOZ2 (ThermoFisher, Cat#PA5-76946), TNNI3 (Santa Cruz, Cat#SC15368), Tubulin (Sigma, Cat#T6199), Pan-QKI(Abcam, Cat#ab126742), QKI-5 (Bethyl, A300-183A), QKI6 (Millipore, Cat#AB9906), and QKI7 (Millipore, Cat#AB9908), respectively, at 1 : 1000 dilution, and corresponding secondary antibodies labeled with horseradish peroxidase (Goat anti-Mouse IgG antibody, ThermoFisher Scientific, G21040; Mouse anti-Rabbit IgG antibody: Santa Cruz, sc-2357) at 1 : 4000 dilution.

Techniques: Expressing, Immunofluorescence, Staining

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: Expressing

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: In Vitro, Labeling, Migration, SDS Page, Molecular Weight, Derivative Assay, Immunoprecipitation, Western Blot

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: RNA Binding Assay, Activity Assay, Binding Assay, Labeling, SDS Page, Western Blot

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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 Ya, Yb, Yc, Yd and Ye 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: Labeling, Mutagenesis, Western Blot, Expressing, RNA Binding Assay

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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 Ya 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: Immunoprecipitation, Derivative Assay, Isolation, Labeling, Mutagenesis, In Vitro, SDS Page, Inhibition

Journal:

Article Title: Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism

doi: 10.1093/emboj/cdg171

Figure Lengend Snippet: 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.

Article Snippet: For kinase reactions using isolated myelin, 500 ng of His-QKI-7 (wt or Y a–e mutant) was bound to Magnetic Ni beads (Qiagene).

Techniques: Expressing, SDS Page, Western Blot, Isolation, Derivative Assay, Standard Deviation