The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5’-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). Maternally expressed gene 3 (MEG3) is a nuclear retained lncRNA found to associate with nuclear speckles. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells, we generated a fluorescently tagged MEG3 transcript that could be detected in real time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Transcription or splicing inactivation conditions, known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with metastasis-associated lung adenocarcinoma (MALAT1) lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions, MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches, we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.

Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select for even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell-lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.

Endogenous gene knock‐in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off‐target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on‐target gene insertions that show the correct sub‐cellular localization of the tagged protein. As such, when searching for cells with on‐target integration using flow cytometry, the off‐target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock‐ins encoding endogenous fluorescent proteins.

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). MEG3 is a nuclear retained lncRNA that was identified to be associated with nuclear speck-les. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells we generated a fluorescently-tagged MEG3 transcript that could be detected in real-time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Conditions under which transcription or splicing were inactive, which are known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with MALAT1 lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.