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Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a large group of modular RNA-binding proteins classified according to their conserved domains. This modular nature, coupled with a large choice of alternative splice variants generates functional diversity. Here, we investigate the biological differences between 40LoVe, its splice variant Samba and its pseudoallele hnRNP AB in neural development. Loss of function experiments lead to defects in neural development with reduction of eye size, which stem primarily from increased apoptosis and reduced proliferation in neural tissues. Despite very high homology between 40LoVe/Samba and hnRNP AB, these proteins display major differences in localization, which appear to be in part responsible for functional differences. Specifically, we show that the 40Love/Samba carboxy-terminal domain (GRD) enables nucleocytoplasmic shuttling behavior. This domain is slightly different in hnRNP AB, leading to nuclear-restricted localization. Finally, we show that shuttling is required for 40LoVe/Samba function in neural development.
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24454782
???displayArticle.pmcLink???PMC3893134 ???displayArticle.link???PLoS One
Figure 1. Alignments and Temporal Expression of 40LoVe, Samba and hnRNP AB.
(A) Alignment of Samba, 40LoVe (GI: 240849376) and hnRNP AB (GI: 148224084) and domain specification. The N-terminus shows high homology with the CBFNT domain identified in the GARG-binding factor A protein [8]. There are two RNA binding domains, the RBD1 containing an RRM1 and the RBD2 containing an RRM2. At the C-terminus of the protein there is a Glycine Rich Domain (GRD). The comparison of hnRNP AB with 40LoVe shows that they have an overall 93% identity at the amino acid level with the differences spread throughout the sequence and indicated here inside the black boxes. (B) Characterization of temporal expression patterns of the three transcripts using RT-PCR. Primers that amplified nucleotides 1-350 (containing the 69 nucleotide deletion in Samba) were used to detect 40LoVe/Samba expression and primers spanning the 5′ UTR to the stop codon were used for RT-PCR detection of hnRNP AB. The first row shows the gel exposed in such a way that allows visualization of both 40LoVe and Samba. Two bands are visible: a strong high molecular weight band corresponding to 40LoVe and a weak lower molecular weight band corresponding to Samba. Rows two and three show 40LoVe and Samba exposed separately so as to clarity expression levels changes and the fourth row shows hnRNP AB levels. 40LoVe, Samba and hnRNP AB are expressed throughout development. Actin was used as a loading control. (C) Western Blot of half embryo equivalent indicates that the three proteins are expressed throughout development and confirms that the expression levels of 40LoVe are higher than those of Samba and that they share similar temporal regulation. Tubulin was used as a loading control. (D) RT-PCR from different regions dissected from a stage 10.5 embryo. 40LoVe/Samba are expressed in all regions of the embryo and hnRNP AB is expressed in a similar manner. The dorsal mesodermal marker Chordin and the neural marker Sox2 were used as controls. (Ds, dorsal region; Vn, Ventral region; An, Animal pole; Vg, Vegetal pole; WE, whole embryo; -RT, samples without reverse transcriptase)
Figure 2. Downregulation of 40LoVe/Samba causes several head and neuronal defects.
(A) 40LoVe/Samba MO1 morphants display generalized head defects with the most prominent being a reduced eye size, eye size, dorsal pigmentation, craniocephalic shape and overall cranial volume. (A�) MO2-injected embryo (24 ng) displayed a milder phenotype compared to MO1-injected embryos. (A��) Co-injection of 80 pg of R40LoVe with 24 ng MO1 partially rescues the phenotype. (A���) Uninjected control embryo. Eye size was measured from embryos injected with MO1, MO2, control morpholino (CoMO), rescued (R2-R40Love co-injected with MO1 and R5-RhnRNPAB-GRD40LoVe co-injected with MO1 as shown in table 1) and control embryos (n = 80�120 for each category). (B) Graph shows that in MO1-injected embryos the eyes were 40% smaller than controls and it was successfully rescued by injection of R40LoVe mRNA (Rescue 2 - eye size 90% of controls). hnRNPAB-GRD40LoVe (Rescue 5) is also able to partially rescue the phenotype. (C) Optical sections from representative whole mount immunostained embryo for acetylated tubulin (red) to reveal neurons. The presence of using GFP (green) indicates the MO-injected side of the embryo (n = 20, three independent experiments). Compared to the uninjected side, the trigeminal nerve (tn), the ophthalmic nerve (on) and the nasocilliary nerves (nc) on the injected side of the embryo (marked with a white circle) are thinner and disorganized. The arrows show the properly formed neurons on the uninjected side of the tadpole. (D) Depth color coded (depth key at bottom of D) 3D reconstruction of optical sections of the trunk of a representative tadpole injected in the animal pole of one out two animal blastomeres with MO1 and then stained using an anti-acetylated tubulin antibody to reveal the axonal projections (n = 30, three independent experiments) 3D reconstruction is shown on the injected side (left) and was then rotated 180 degrees and shown on the uninjected side (right). Motor neuron projections rising from the spinal cord on the injected side of the embryo are absent or short in contrast to the un-injected side. (E) Whole mount in situ hybridization using N-tubulin (Ntub) and Sox10 show that the neural tissues are defined normally in MO1-injected embryos. However, tissues such as the cranial sensory ganglia and the branchial arches are misshapen and did not migrate normally (arrowheads) and expression levels Ntub and Sox 10 appear reduced in the injected side. (F) RT-PCR experiments confirm a reduction of neural marker expression in MO1 injected embryos. Actin was used as a loading control.
Figure 3. 40LoVe/Samba are required for neuronal cell survival.
(A) Hoechst (cyan) and Histone H3[p Ser10] (magenta) staining reveals mitotic cells of a representative embryo injected with MO1 in one out of two animal blastomeres at the two cell stage. The images show that fewer dividing cells are present in the MO1 injected side of the embryo compared to the uninjected side of the same embryo. The graph shows quantification from 15 embryos from two independent experiments. (B) Whole mount in situ hybridization using the specific eye marker XrX1 shows reduced expression in the eye on the injected side compared to the control side. TUNEL staining reveals increased apoptosis in the eye and other head structures in MO1-injected side of the embryo compared to the uninjected side of the same embryo.
Figure 4. 40LoVe/Samba, hnRNP AB, deletion mutant and fusion construct localization in XL177 cells.
In each figure cells were co-electroporated with a GFP-tagged version of the protein indicated on the left (green) and Histone-RFP to label the nucleus (red). The first column shows the merged image of both the indicated construct (green) and histone (red), while the second column shows the construct localization alone. The third column shows the intensity profile for each cell along the red line shown in the second column. Red and blue lines represent the boundaries of the nucleus of each cell. (A�B) Samba and 40LoVe are localized both in the nucleus and the cytosol. (C) hnRNP AB is exclusively nuclear. (D) 40LoVe-ABN localizes like 40LoVe, suggesting that the N-terminus of the protein is not responsible for nuclear retention of hnRNP AB. (E) ABδGRD loses the strictly nuclear localization of hnRNP AB, indicating that the GRD domain is necessary for the exclusively nuclear localization of hnRNP AB. (F) 40LoVeGRDAB localizes exactly like hnRNP AB, showing that the GRD domain of hnRNP AB is sufficient for nuclear retention. (G) hnRNP AB-GRD40LoVe localizes like 40LoVe, confirming that the hnRNP AB GRD is necessary for nuclear retention of hnRNP AB and that that the few amino acid differences between the GRD domains of the two proteins are responsible for their differences in localization.
Figure 5. 40LoVe/Samba shuttles from the nucleus to the cytosol and the GRD domain is both necessary and sufficient for this process.
(A) Intensity coded still images (intensity scale is shown below the set of images) from a time lapse recording of a FRAP experiment in a cell co-expressing GFP-Samba and Histone RFP. The area outlined in blue (the nucleus) was bleached and the GFP signal intensity was monitored over time both in the nucleus (blue outline) and the cytosol (white outline). The graph shows the fluorescence intensity in the nucleus and the cytosol. The intensity difference between the two compartments is nearly extinguished after about 40 minutes. After 40 minutes the nuclear signal has recovered while the cytsosolic signal has decreased showing that cytosolic protein moved into the nucleus and suggesting that bleached molecules are exiting the nucleus and moving into the cytosol. (B) Intensity coded still images (intensity scale is shown below the set of images) from a time lapse recording of a FLIP experiment. When the cytosol (white outline) is repeatedly bleached (red circle), signal intensity in the nucleus (outline) diminishes gradually confirming that molecules in the nucleus are moving into the cytosol. The graph shows the fluorescence intensity in the nucleus (blue outline) and the cytosol (white outline). (C) Intensity coded still images (intensity scale is shown below the set of images) from a time lapse recording of a FRAP experiment from a GFP-SambaδGRD expressing cell. Unlike full length Samba the signal intensity in the nucleus fails to recover while the signal in the cytosol also fails to decrease suggesting that SambaδGRD does not shuttle from the cytoplasm to the nucleus. The graph shows the fluorescence intensity in the nucleus (outlined blue) and cytosol (outlined white) over time. The intensity difference between the two compartments is reduced but fails to be extinguished even after about 40 minutes. (D) Intensity-coded still images (intensity scale is shown below the set of images) from a time lapse recording of a FRAP experiment from an hnRNPAB-GRD40LoVe expressing cell. Nuclear signal recovers while cytosolic signal decreases over time in a similar fashion to what is observed with GFP-Samba suggesting that the GRD domain of 40LoVe is sufficient to confer shuttling from the nucleus to the cytoplasm. The graph was generated from measurements in the cytosol (outlined white) circle and the nucleus (outlined blue). The intensity difference between the two compartments is nearly extinguished after about 40 minutes showing similar dynamics to GFP-Samba (A).
Figure S1.
Morpholino downregulation of 40LoVe/Samba. (A) Western Blot of half embryo equivalent injected with 60 pg of surrogate Samba-flag or hnRNP AB alone or co-injected with 8 ng, 16 ng and 24 ng MO1 as indicated. MO1 effectively downregulates both Samba/40LoVe and hnRNP AB. (B) Western Blot of half embryo equivalent injected with 60 pg of surrogate hnRNP AB or 40LoVe alone or co-injected with 8 ng, 16 ng and 24 ng MO1 as indicated. MO2 fails to downregulate hnRNP AB but downregulates 40LoVe/Samba. (C) Western Blot of half embryo equivalent injected with 12 ng, 24 ng and 30 ng MO1 shows that MO1 can effectively downregulate endogenous 40LoVe. Tubulin was used as a loading control. (D) Immunofluorescence experiments using the 40LoVe antibody confirm that MO1 down-regulates the endogenous protein. mGFP was used as a linage tracer of MO1 injected cells.
Figure S2.
Mutants generated for the determination of the protein domains responsible for the differences in localization between the three proteins. 40LoVe-ABN was constructed using the N-terminus of hnRNP AB form start to nucleotide 270/amino acid 90 and the rest of 40LoVe protein from nucleotide 273/amino acid 91 to the stop codon. ABδGRD is hnRNP AB with a deleted c-terminus form nucleotide 630/amino acid 210 with an added stop codon. 40LoVeGRDAB was constructed with 40LoVe from start codon to nucleotide 666/amino acid 222 fused with hnRNP AB from nucleotide 633/amino acid 221 to stop codon. hnRNP AB-GRD40LoVe was constructed with hnRNP AB from start codon to nucleotide 630/amino acid 210 fused with 40LoVe from amino acid 690/amino acid 230 to stop codon. All constructs are fused with a FLAG-tag at the C-terminus.
Figure S3.
Alignment of the GRD domains of 40LoVe/Samba, hnRNP AB and the human hnRNP D (GI: 51477711). DNS is the 19 amino acid sequence highlighted in turquoise that has been shown to be responsible for shuttling in hnRNP D. Two out of the three differences in the GRD domain between 40LoVe/Samba and hnRNP AB are located in this 19 amino acid sequence. The yellow highlighted amino acid is the one likely responsible for differences in localization between 40LoVe/Samba and hnRNP AB. This amino acid is an Asparagine in hnRNP D and hnRNP AB, which both are strictly nuclear, but it's a Serine in 40LoVe/Samba which show both nuclear and cytosolic localization.
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