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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 II. Higher magnification SIM images of two of the representative single paraspeckles stained with the Neat1_mid and the Neat1_5′ probe (A), the Neat1_mid and the Neat1_3′ probe (B), the Neat1_mid and the Neat1_5′+3′ probe (C), and the Neat1_3′ and the Neat1_ 5′ probe (D). Intensity profiles along the dashed lines (a and b) are shown in the graphs. Note that the middle region of Neat1 is centrally located, and the 5′ and the 3′ regions are distributed in a complementary manner along the shell of the paraspeckle spheres. Bar, 100 nm.
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fig2: Core-shell arrangement of Neat1 in paraspeckle spheres II. Higher magnification SIM images of two of the representative single paraspeckles stained with the Neat1_mid and the Neat1_5′ probe (A), the Neat1_mid and the Neat1_3′ probe (B), the Neat1_mid and the Neat1_5′+3′ probe (C), and the Neat1_3′ and the Neat1_ 5′ probe (D). Intensity profiles along the dashed lines (a and b) are shown in the graphs. Note that the middle region of Neat1 is centrally located, and the 5′ and the 3′ regions are distributed in a complementary manner along the shell of the paraspeckle spheres. Bar, 100 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 II. Higher magnification SIM images of two of the representative single paraspeckles stained with the Neat1_mid and the Neat1_5′ probe (A), the Neat1_mid and the Neat1_3′ probe (B), the Neat1_mid and the Neat1_5′+3′ probe (C), and the Neat1_3′ and the Neat1_ 5′ probe (D). Intensity profiles along the dashed lines (a and b) are shown in the graphs. Note that the middle region of Neat1 is centrally located, and the 5′ and the 3′ regions are distributed in a complementary manner along the shell of the paraspeckle spheres. Bar, 100 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Core-shell arrangement of Neat1 in paraspeckle spheres II. Higher magnification SIM images of two of the representative single paraspeckles stained with the Neat1_mid and the Neat1_5′ probe (A), the Neat1_mid and the Neat1_3′ probe (B), the Neat1_mid and the Neat1_5′+3′ probe (C), and the Neat1_3′ and the Neat1_ 5′ probe (D). Intensity profiles along the dashed lines (a and b) are shown in the graphs. Note that the middle region of Neat1 is centrally located, and the 5′ and the 3′ regions are distributed in a complementary manner along the shell of the paraspeckle spheres. Bar, 100 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.