Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs.To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (linkers of the nucleoskeleton and the cytoskeleton). Previous studies have demonstrated that the testis-specific SUN domain protein SUN4 is a vital player in the directed shaping of the spermatid nucleus. However, its molecular properties relating to this crucial function have remained largely unknown and controversial data for the organization and orientation of SUN4 within the spermatid nuclear envelope have been presented so far. Here we have re-evaluated this issue in detail and show robust evidence that SUN4 is integral to the inner nuclear membrane, sharing a classical SUN domain protein topology. The C-terminal SUN domain of SUN4 localizes to the perinuclear space, while the N-terminus is directed to the nucleoplasm, interacting with the spermiogenesis-specific lamin B3. We found that SUN4 forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Together, our results contribute to a better understanding of the specific function of SUN4 at the spermatid nucleo-cytoplasmic junction and the process of sperm-head formation.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (linkers of the nucleoskeleton and the cytoskeleton). Previous studies have demonstrated that the testis-specific SUN domain protein Sun4 is a vital player in spermatogenesis, critically involved in the directed shaping of the spermatid nucleus. Its molecular properties relating to this crucial function, however, have remained largely unknown. Previous studies presented quite controversial data for the general organization and orientation of Sun4 within the spermatid nuclear envelope. In the present study, we have re-evaluated this issue in detail and present new robust data on the Sun4 topology and its interactions at the nucleo-cytoplasmic junction. We identified Sun4 as an integral protein of the inner nuclear membrane, sharing a classical SUN domain protein topology. Similar to other SUN domain proteins, the C-terminal SUN domain of Sun4 locates to the perinuclear space and the N-terminus is directed to the nucleoplasm, where it interacts with the spermiogenesis specific Lamin B3. We found that Sun4 in its natural environment forms heteromeric assemblies with Sun3 and, beyond this, it is crucially involved in the regulation of Sun3 expression. Together, our results contribute to a better understanding of the specific function of Sun4 at the spermatid nucleo-cytoplasmic junction and the entire process of sperm-head formation.Summary statementIn our current study, we have analyzed in detail the biochemical and dynamic properties of the testis-specific SUN domain protein Sun4 and we provide novel insights into its interaction behavior at the spermatid nucleo-cytoplasmic junction.