Journal: Scientific Reports

Article Title: Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

doi: 10.1038/s41598-024-79409-9

Figure Lengend Snippet: hnRNPAB X2 is a novel cytoplasmic splice isoform of hnRNPAB that is enriched in L-bodies. ( a ) Stage II oocytes were microinjected with Cy5-labeled LE RNA (magenta, a′) and immunostained for endogenous hnRNPAB (green, a) using antibodies raised against Xenopus hnRNPAB . The overlap is shown in a′′. ( b ) Cy5-labeled LE RNA (magenta, b′) was microinjected into stage II oocytes expressing mCherry-tagged canonical hnRNPAB, as detected by immunostaining with anti-RFP (green, b). The overlap is shown in b′′. ( c ) Schematics of canonical hnRNPAB, hnRNPAB X2 and hnRNPABΔPY. C-terminal sequence is shown below each, with the PY NLS indicated in red, the protein coding sequence (CDS) in gray, and the 3′UTR shown in orange. ( d ) RNA isolated from stage II oocytes was used to measure the relative expression of hnRNPAB X2 compared to canonical hnRNPAB by qPCR. ΔCt values were calculated normalizing to refence gene vg1 (* indicates p < 0.05 by T-test). Error bars represent standard deviation of the mean, n = 3. ( e ) Cy5-labeled LE RNA (magenta, e′) was microinjected into stage II oocytes expressing mCherry-tagged hnRNPAB X2, as detected by immunostaining with anti-RFP (green, e). The overlap is shown in e′′. ( f ) Cy5-labeled LE RNA (magenta, e′) was microinjected into stage II oocytes expressing mCherry-tagged hnRNPABΔPY, as detected by immunostaining with anti-RFP (green, e). The overlap is shown in e′′. Confocal sections (a-b, e-f) are shown with the vegetal hemisphere at the bottom; scale bars = 100 μm.

Article Snippet: Fluorescently labeled vg1 LE RNA (Gautreau et al., 1997) was generated from linearized pSP73-2×135 using the MEGAscript T7 transcription kit (Ambion) in the presence of 250 nM Cy5-UTP (Fisher Scientific, PA55026). mCherry (mCh)-tagged protein-coding mRNAs were generated using the mMessage Machine transcription kit from the following linearized plasmids: pSP64:mCh-hnRNPAB-X2, pSP64:mCh-RBD-X2, pSP64:mCh-IDR-X2, pSP64:mCh, pSP64:mCh-hnRNPAB-FL, pSP64:mCh-hnRNPABΔPY, pSP64:mCh-PTBP3 and pSP64:mCh-PTBP3 + IDR. mRNAs and Cy-labeled RNAs were co-microinjected at 500 nM at a volume of 2 nL per oocyte.

Techniques: Labeling, Expressing, Immunostaining, Sequencing, Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

doi: 10.1038/s41598-024-79409-9

Figure Lengend Snippet: hnRNPAB X2 enriches in L-bodies through its RBD and IDR. ( a ) Schematics of domain constructs. ( b – e ) Stage II oocytes expressing ( b ) mCh-hnRNPAB X2, ( c ) mCh-RBD, ( d ) mCh-IDR, or ( e ) free mCh (green; detected by anti-RFP IF) were co-microinjected with Cy5 LE RNA (magenta, b′-e′) to label L-bodies. Colocalization (white) is shown in the merged confocal images (b′-e′). Scale bars = 100 μm. ( f ) L-body enrichment was quantitated by measuring the fraction of total protein fluorescence localized to the vegetal cortical region. Shown are relative levels of L-body enrichment for hnRNPAB X2 (green, set to 1.0 ± 0.075), RBD (blue, 0.71 ± 0.076), IDR (orange, 0.75 ± 0.059), and mCherry (red, 0.38 ± 0.034). n = 24 oocytes from four biological replicates; error bars represent standard error of the mean, **** indicates p < 0.0001, *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, and ns indicates not significant ( p > 0.05). Statistics shown are an ordinary one-way ANOVA followed by Tukey’s multiple comparisons.

Article Snippet: Fluorescently labeled vg1 LE RNA (Gautreau et al., 1997) was generated from linearized pSP73-2×135 using the MEGAscript T7 transcription kit (Ambion) in the presence of 250 nM Cy5-UTP (Fisher Scientific, PA55026). mCherry (mCh)-tagged protein-coding mRNAs were generated using the mMessage Machine transcription kit from the following linearized plasmids: pSP64:mCh-hnRNPAB-X2, pSP64:mCh-RBD-X2, pSP64:mCh-IDR-X2, pSP64:mCh, pSP64:mCh-hnRNPAB-FL, pSP64:mCh-hnRNPABΔPY, pSP64:mCh-PTBP3 and pSP64:mCh-PTBP3 + IDR. mRNAs and Cy-labeled RNAs were co-microinjected at 500 nM at a volume of 2 nL per oocyte.

Techniques: Construct, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

doi: 10.1038/s41598-024-79409-9

Figure Lengend Snippet: hnRNPAB X2 dynamically associates with L-bodies via its RBD and IDR. ( a ) Shown is an image of the vegetal cytoplasm of a stage II oocyte expressing mCh-hnRNPAB X2. FRAP was conducted such that an individual L-body was partially bleached (a′), to allow for recovery (a′′) both from within the L-body and from its environment. The 10 μm 2 ROI is indicated by a white box; scale bar = 10 μm. ( b ) Stage II oocytes were microinjected with mCherry (mCh), mCh-hnRNPAB X2, mCh-RBD or mCh-IDR RNA to express the mCh-tagged proteins, along with Cy5 LE RNA to mark L-bodies. Normalized FRAP recovery curves are shown ( n = 21 oocytes per fusion protein); error bars represent standard error of the mean. Measurements were taken at 5 s intervals over 100 iterations. ( c ) Plateau values (% mobile fraction) are shown for mCh-hnRNPAB X2 (green, 96%±0.013), mCh-RBD (blue, 98%±0.014), mCh-IDR (orange, 98%±0.002), and mCherry (red, 104%±0.29). Error bars represent standard error of the mean. Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons; ns indicates not significant ( p > 0.05) and * indicates p = 0.04. ( d ) T 1/2 measurements are shown for mCh-hnRNPAB X2 (green, 30.1s ± 3.4), mCh-RBD (blue, 9.3s ± 0.4), mCh-IDR (orange, 10.1s ± 0.6), and mCherry (red, 6.0s ± 1.6), measured as the average time at which the protein recovers half of its plateau value. Error bars represent standard error of the mean. Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons; ns indicates not significant ( p > 0.05), *** indicates p < 0.001, **** indicates p < 0.0001.

Article Snippet: Fluorescently labeled vg1 LE RNA (Gautreau et al., 1997) was generated from linearized pSP73-2×135 using the MEGAscript T7 transcription kit (Ambion) in the presence of 250 nM Cy5-UTP (Fisher Scientific, PA55026). mCherry (mCh)-tagged protein-coding mRNAs were generated using the mMessage Machine transcription kit from the following linearized plasmids: pSP64:mCh-hnRNPAB-X2, pSP64:mCh-RBD-X2, pSP64:mCh-IDR-X2, pSP64:mCh, pSP64:mCh-hnRNPAB-FL, pSP64:mCh-hnRNPABΔPY, pSP64:mCh-PTBP3 and pSP64:mCh-PTBP3 + IDR. mRNAs and Cy-labeled RNAs were co-microinjected at 500 nM at a volume of 2 nL per oocyte.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

doi: 10.1038/s41598-024-79409-9

Figure Lengend Snippet: The hnRNPAB X2 IDR acts to stabilize proteins in L-bodies. ( a ) The hnRNPAB X2 IDR was fused to the C-terminus of PTBP3 (PTBP3+X2IDR) and tagged with mCherry (mCh). ( b ) Stage II oocytes were microinjected with mCh-PTBP3 RNA to express the encoded protein (green, detected by anti-RFP IF) along with Cy5 LE RNA to label L-bodies (magenta, b′). The merge is shown in b′′; scale bar = 100 μm. ( c ) Stage II oocytes were microinjected with mCh-PTBP3+IDR to express the encoded protein (green, detected by anti-RFP IF) along with Cy5 LE RNA (magenta, c′) to label L-bodies. The merge is shown in c′′; scale bar = 100 μm. ( d ) Stage II oocytes were microinjected with RNA encoding PTBP3 or PTBP3+X2IDR, along with Cy5 LE RNA to label L-bodies. Normalized FRAP recovery curves are shown ( n = 21 oocytes); error bars represent standard error of the mean. Measurements were taken at 5 s intervals over 100 iterations. ( e ) Average percent mobile fractions are shown for mCh-PTBP3 (green, 40%±0.88) and mCh-PTBP3+X2IDR (blue, 20%±2.31). Error bars represent standard error of the mean and ** indicates p < 0.01. Statistics shown are an Ordinary one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Fluorescently labeled vg1 LE RNA (Gautreau et al., 1997) was generated from linearized pSP73-2×135 using the MEGAscript T7 transcription kit (Ambion) in the presence of 250 nM Cy5-UTP (Fisher Scientific, PA55026). mCherry (mCh)-tagged protein-coding mRNAs were generated using the mMessage Machine transcription kit from the following linearized plasmids: pSP64:mCh-hnRNPAB-X2, pSP64:mCh-RBD-X2, pSP64:mCh-IDR-X2, pSP64:mCh, pSP64:mCh-hnRNPAB-FL, pSP64:mCh-hnRNPABΔPY, pSP64:mCh-PTBP3 and pSP64:mCh-PTBP3 + IDR. mRNAs and Cy-labeled RNAs were co-microinjected at 500 nM at a volume of 2 nL per oocyte.

Techniques:

Journal: bioRxiv

Article Title: Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

doi: 10.1101/585679

Figure Lengend Snippet: (A) msp300 mRNA co-precipitates with Syp. Quantification of RT-qPCR data shows high enrichment of msp300 relative to the non-binding control rp49 (mean ± sem, N=3 IPs). (B-D) Representative confocal microscopy image of msp300 smFISH and Syp-GFP signal at the larval NMJ, max z projection. (E-G) A single confocal slice of the same image, showing that msp300 transcripts co-localise with Syp containing RNP granules within the resolution limit of the system. (H) Schematic of Cy5-labeled msp300 RNA injection experiment in larval NMJ preparation to test its association with Syp. (I-J) Representative live images of Syp-GFP in a larval muscle injected with Cy5-labelled msp300 mRNA. (K) A bright field image of an injected muscle indicates that the muscle still appears healthy 60 minutes after the injection. (L-N) Region of interest (from box in I) where ccRICS data were acquired. Each image is a sum projection of 50 images acquired in photon-counting mode. (O-Q) Autocorrelation function acquired from the images in L-N. (R-T) 3D plots of the spatial autocorrelation function with the relative amplitude and residuals after fitting with the RICS equation showing that ~12% of molecular complexes contain both Syp protein and msp300 mRNA.

Article Snippet: Cy5-labelled msp300 RNA (Cy5-UTP) was pressure injected into Drosophila larval muscles using pre-fabricated glass capillary tips (0.5μm inner diameter, 1.0 μm outer diameter; Eppendorf Femptotips).

Techniques: Quantitative RT-PCR, Binding Assay, Confocal Microscopy, Labeling, Injection