Posturas epistemológicas respecto a las ciencias

3.1.1. Dual RNA imaging in neurons

Fusion of fluorescent proteins (FPs) to the MS2 or the λN22 peptide, enables the visualization of single RNAs in neurons. The modular character of AFP-based labeling provides all prerequisites to track several specific RNAs at the same time. This has proven possible for the first time by tagging two distinct mRNA in yeast in real time (Lange et al. 2008).

In order to get insight into distribution and transport kinetics of several specific RNAs in one cell we are taking advantage of these viral RNA reporters for the tagging of neuronal RNAs of interest. We constructed a fusion protein yielding either the λN22 peptide or the MS2 peptide, 3 copies of EGFP or mKO2, respectively, and the SV40 nuclear localization signal (NLS) driven by the CMV promoter. Rat hippocampal neurons DIV10 transiently transfected with both RNA labels were stimulated using 40 mM KCl for 2 h (Figure 23). We tagged several mRNAs including CaMKIIα with the corresponding aptamers boxB or MS2. Nuclear export efficiency for the single labeling systems were determined by counting cells with exclusive nuclear versus nuclear and dendritic fluorescence signal (Figure 23B). In the presence of MS2-tagged CaMKIIα the MS2- 3xmKO2-NLS reporter was exported from the nucleus in 43% of the neurons compared to 34% in the absence of any tagged RNA. For the λN22-3xEGFP-NLS construct the difference was 38% with CaMKIIα-boxB cotransfection compared to 35% with the reporter construct only. Double labeling using the MS2- and the λN22-systems showed mainly nuclear localized fluorescence of both reporter without any transiently expressed mRNA (Figure 23C). Serving as a direct comparison of the two labeling systems, 5 copies of the boxb and 24 copies of MS2 aptamers were cloned between the CaMKIIα ORF and the 5’UTR or 3’UTR. mKO2 and EGFP signals were detected at the dendrite with EGFP showing a rather proximal and granular pattern compared to the mKO2-tagged MS2 peptide (Figure 23D). Expression of CaMKIIα tagged with boxB only resulted in dendritic localization of λN22-3xEGFP-NLS but nuclear red fluorescence (Figure 23E). This indicates the lack of interference of both labeling systems.

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Figure 23 MS2 and λN22 labeling in rat hippocampal neurons. (A) RNA label constructs used for transfection. (B) Quantification of RNA label nuclear export in rat hippocampal neurons, DIV10. Percentage of cells with somatic signal of the fluorescent labels. Hatched bars indicate cells transfected with the reporter only, filled bars represent neurons expressing both label and tagged RNA. MS system (red), λN22 system (green). (C-E) Confocal images of rat hippocampal neurons, DIV10, transiently transfected using lipofectamine. Neurons expressing MS2-3xmKO2-NLS and λN22- 3xEGFP-NLS only (C), with boxB- and MS2-tagged CaMKIIα (D) or CaMKIIα-boxB (E).

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3.1.2. NMDAR1 splicing reporter

The MS2- and λN22-RNA labeling systems were used to establish a reporter for NMDAR1 alternative splicing of exon 21. We constructed a splice reporter based on the established exon 21 minigene (Wenqing Zhang et al. 2002) (J Han 2005). The MS2 tag was inserted between the 5`UTR and exon20 followed by an intron and a boxB-tagged exon 21, another intron, exon 22 and the 3`UTR (Figure 24). Both alternative splice variants are tagged by MS2 but only the variant bearing exon 21 keeps the boxB aptamer after intron removal.

Figure 24 NMDAR1 splicing reporter strategy. The artificial NMDAR1 pre-mRNA reporter construct contains both RNA tags that can be bound by MS2-3xmKO2-NLS and λN22-3xEGFP-NLS. After splicing, exon 21 bearing the boxB tag at the 3`end is retained or spliced depending on splice site selection.

The NMDAR1 splicing reporter RNA and λN22-3xEGFP-NLS were transiently expressed in hippocampal neurons, DIV10. We observed dendritic granular labeling that was further characterized by costaining with different markers. The λN22 label did not colocalize with lysotracker, the ER or P-body marker DCP1. NMDAR1 signal partially overlapped with S6 ribosomal protein and with FMRP in stress granules (Figure 25).

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Figure 25 NMDAR1 splice reporter granule characterization. (A) Confocal images of rat hippocampal neuron dendritic branches expressing NMDAR1 RNA reporter and λN22-3xEGFP-NLS. Neurons were either fixed and costained with antibodies for DCP1, S6 ribosomal protein and FMRP using a Cy3 secondary or antibody. Alternatively, cells were supplied with lysotracker or the ER-targeted mKO2 was coexpressed. (B) Schematic of NMDAR1 reporter association with dendritic structures.

The NMDAR1 splicing RNA construct and both, the MS2 and the λN22 labels were transfected into hippocampal neurons. Confocal imaging revealed different distribution of fluorescence emission of the two fluorescent proteins (Figure 26A). Double-labeled granules correspond to locations of NMDAR1 reporter pre-mRNA or exon 21-retained splice variant. Regions with EGFP but no mKO2 emission indicated the presence of exon 21-spliced NMDAR1 reporter RNA. A program was written by Friedrich Förstner that measures emission intensity ratio IR/G of mKO2 and EGFP and reassigns red colour for IR/G>1 and green colour for IR/G<1 (Figure 26B). mKO2 and EGFP emission intensities were plotted as a function of distance from the nucleus by defining a region within the nucleus as the r=0 (Figure 26C). Different neurons and dendrites show varying distribution of the two reporter signals.

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Figure 26 NMDAR1 exon21 alternative splice variant imaging in hippocampal neurons. (A) NMDAR (NR1) splicing RNA reporter construct and the MS2-3xmKO2-NLS (red) and λN22-3xEGFP-NLS (green) were transiently expressed in hippocampal neurons, DIV10 and cells were stimulated by 40 mM KCl for 2 h. (B) Red and green colors were assigned to regions of IR/G>1 and IR/G<1, respectively.

(C) Magnifications from the fluorescence image (A, white box) of both channels with a white circle labeling the region at the proximal dendrite defined as r=0. EGFP or mKO2 emission intensity are dependent on the distance to the predefined region.