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Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

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ABSTRACT

Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1. Using structural illumination microscopy, West et al. analyze the organization of paraspeckles at the submicron scale and show that paraspeckle proteins are arranged around bundles of Neat1, forming core-shell spheroidal structures dependent on the RNA binding protein Fus.

No MeSH data available.


Core-shell arrangement of Neat1 in paraspeckle spheres I. (A) Schematic diagrams of the positions of FISH probes that detect differential regions of Neat1. (B and C) Simultaneous detection of the middle and 3′ regions of Neat1 using a conventional epifluorescence microscope (Conventional) and SIM. (D) The same FISH image detected with the converse combination of secondary antibodies as in C. (E) Comparisons of the differential distribution of each Neat1 region in the paraspeckle spheres. Note that the middle region is located in the core of the paraspeckles, whereas the 5′ and the 3′ regions are located peripherally. Asterisks indicate the position of the putative transcription site detected with the Neat1_tail probe. (F) Paraspeckles with a sausage-like shape that were occasionally detected in the corpus luteal cells. (G) Histogram of paraspeckles with different shapes (n = 187). Bars: (B) 5 µm; (C–F) 500 nm.
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fig1: Core-shell arrangement of Neat1 in paraspeckle spheres I. (A) Schematic diagrams of the positions of FISH probes that detect differential regions of Neat1. (B and C) Simultaneous detection of the middle and 3′ regions of Neat1 using a conventional epifluorescence microscope (Conventional) and SIM. (D) The same FISH image detected with the converse combination of secondary antibodies as in C. (E) Comparisons of the differential distribution of each Neat1 region in the paraspeckle spheres. Note that the middle region is located in the core of the paraspeckles, whereas the 5′ and the 3′ regions are located peripherally. Asterisks indicate the position of the putative transcription site detected with the Neat1_tail probe. (F) Paraspeckles with a sausage-like shape that were occasionally detected in the corpus luteal cells. (G) Histogram of paraspeckles with different shapes (n = 187). Bars: (B) 5 µm; (C–F) 500 nm.

Mentions: To gain insight into the molecular mechanism and function of paraspeckles, we examined their fine structure using SIM and compared the spatial relationship between different regions of Neat1_2 (hereafter, Neat1) and paraspeckle proteins in detail. For this analysis, we used primary cultures of corpus luteal cells expressing luteal marker genes (Fig. S1), as the physiological function of paraspeckles in this cell type has been well documented in Neat1 KO mice (Nakagawa et al., 2014). First, we performed FISH and simultaneously detected the middle and the 3′ regions of Neat1 using probes that specifically detected each region (Fig. 1 A). The signals obtained using these probes largely overlapped when using a conventional epifluorescence microscope (Fig. 1, B and C). However, a single focus SIM observation clearly revealed a differential arrangement of the two regions of Neat1, with the centrally located middle region surrounded by the 3′ region located peripherally, forming a core-shell spheroidal structure (Fig. 1 C, Fig. 2 B, and Fig. S3). The characteristic core-shell organization of Neat1 was consistent with previous electron microscopy observations (Souquere et al., 2010), indicating the validity of SIM for the observation and study of nuclear bodies. We also confirmed the core-shell structure using an inverse combination of fluorescent dyes (Fig. 1 D), suggesting that the layered organization of the two signals was not an artifact caused by the differential diffraction limits of the two different wavelengths of the light.


Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization
Core-shell arrangement of Neat1 in paraspeckle spheres I. (A) Schematic diagrams of the positions of FISH probes that detect differential regions of Neat1. (B and C) Simultaneous detection of the middle and 3′ regions of Neat1 using a conventional epifluorescence microscope (Conventional) and SIM. (D) The same FISH image detected with the converse combination of secondary antibodies as in C. (E) Comparisons of the differential distribution of each Neat1 region in the paraspeckle spheres. Note that the middle region is located in the core of the paraspeckles, whereas the 5′ and the 3′ regions are located peripherally. Asterisks indicate the position of the putative transcription site detected with the Neat1_tail probe. (F) Paraspeckles with a sausage-like shape that were occasionally detected in the corpus luteal cells. (G) Histogram of paraspeckles with different shapes (n = 187). Bars: (B) 5 µm; (C–F) 500 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037409&req=5

fig1: Core-shell arrangement of Neat1 in paraspeckle spheres I. (A) Schematic diagrams of the positions of FISH probes that detect differential regions of Neat1. (B and C) Simultaneous detection of the middle and 3′ regions of Neat1 using a conventional epifluorescence microscope (Conventional) and SIM. (D) The same FISH image detected with the converse combination of secondary antibodies as in C. (E) Comparisons of the differential distribution of each Neat1 region in the paraspeckle spheres. Note that the middle region is located in the core of the paraspeckles, whereas the 5′ and the 3′ regions are located peripherally. Asterisks indicate the position of the putative transcription site detected with the Neat1_tail probe. (F) Paraspeckles with a sausage-like shape that were occasionally detected in the corpus luteal cells. (G) Histogram of paraspeckles with different shapes (n = 187). Bars: (B) 5 µm; (C–F) 500 nm.
Mentions: To gain insight into the molecular mechanism and function of paraspeckles, we examined their fine structure using SIM and compared the spatial relationship between different regions of Neat1_2 (hereafter, Neat1) and paraspeckle proteins in detail. For this analysis, we used primary cultures of corpus luteal cells expressing luteal marker genes (Fig. S1), as the physiological function of paraspeckles in this cell type has been well documented in Neat1 KO mice (Nakagawa et al., 2014). First, we performed FISH and simultaneously detected the middle and the 3′ regions of Neat1 using probes that specifically detected each region (Fig. 1 A). The signals obtained using these probes largely overlapped when using a conventional epifluorescence microscope (Fig. 1, B and C). However, a single focus SIM observation clearly revealed a differential arrangement of the two regions of Neat1, with the centrally located middle region surrounded by the 3′ region located peripherally, forming a core-shell spheroidal structure (Fig. 1 C, Fig. 2 B, and Fig. S3). The characteristic core-shell organization of Neat1 was consistent with previous electron microscopy observations (Souquere et al., 2010), indicating the validity of SIM for the observation and study of nuclear bodies. We also confirmed the core-shell structure using an inverse combination of fluorescent dyes (Fig. 1 D), suggesting that the layered organization of the two signals was not an artifact caused by the differential diffraction limits of the two different wavelengths of the light.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1. Using structural illumination microscopy, West et al. analyze the organization of paraspeckles at the submicron scale and show that paraspeckle proteins are arranged around bundles of Neat1, forming core-shell spheroidal structures dependent on the RNA binding protein Fus.

No MeSH data available.