Single molecule RNA localization and translation in the mammalian oocyte and embryo

AbstractWhile the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect and quantify single molecule RNA localization patterns in skeletal myofibers, and uncover a critical role for directed transport of RNPs in muscle. We find that RNAs localize and are translated along sarcomere Z-disks, dispersing tens of microns from progenitor nuclei, regardless of encoded protein function. We find that directed transport along the lattice-like microtubule network of myofibers becomes essential to achieve this localization pattern as muscle development progresses; disruption of this network leads to extreme accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that global active RNP transport may be required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms of gene regulation, with consequences for myopathies caused by perturbations to RNPs or microtubules.

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FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences

10.1101/784728 ◽

2019 ◽

Cited By ~ 2

Author(s):

Raeann Goering ◽

Laura I. Hudish ◽

Bryan B. Guzman ◽

Nisha Raj ◽

Gary J. Bassell ◽

...

Keyword(s):

Single Molecule ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Rna Localization ◽

Null Mouse ◽

Rna Sequences ◽

Local Protein Synthesis ◽

Rna Molecules ◽

G Quadruplex

ABSTRACTThe sorting of RNA molecules to distinct subcellular locations facilitates the activity of spatially restricted processes through local protein synthesis. This process affects thousands of transcripts yet precisely how these RNAs are trafficked to their destinations remains generally unclear. Here we have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these FMRP RNA localization targets contain a large enrichment of G-quadruplex sequences, particularly in their 3′ UTRs, suggesting that FMRP recognizes these sequences to promote the localization of transcripts that contain them. Fractionation of neurons derived from human Fragile X Syndrome patients revealed a high degree of conservation in the identity of FMRP localization targets between human and mouse as well as an enrichment of G-quadruplex sequences in human FMRP RNA localization targets. Using high-throughput RNA/protein interaction assays and single-molecule RNA FISH, we identified the RGG domain of FMRP as important for both interaction with G-quadruplex RNA sequences and the neuronal transport of G-quadruplex-containing transcripts. Finally, we used ribosome footprinting to identify translational regulatory targets of FMRP. The translational regulatory targets were not enriched for G-quadruplex sequences and were largely distinct from the RNA localization targets of FMRP, indicating that the two functions can be biochemically separated and are mediated through different target recognition mechanisms. These results establish a molecular mechanism underlying FMRP-mediated neuronal RNA localization and provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

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Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-011520-104955 ◽

2020 ◽

Vol 89 (1) ◽

pp. 159-187 ◽

Cited By ~ 4

Author(s):

Hanae Sato ◽

Sulagna Das ◽

Robert H. Singer ◽

Maria Vera

Keyword(s):

Fluorescence Microscopy ◽

Single Molecule ◽

Rna Processing ◽

Functional Organization ◽

Nuclear Architecture ◽

Rna Localization ◽

Rna Molecules ◽

Cell Functions ◽

Dna And Rna ◽

Multicolor Fluorescence

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

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The method of replication affects metal film granularity and resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155517 ◽

1989 ◽

Vol 47 ◽

pp. 708-709

Author(s):

George C. Ruben

Keyword(s):

Electron Beam ◽

High Resolution ◽

Film Thickness ◽

Single Molecule ◽

Metal Film ◽

Vertical Surface ◽

High Resolution Imaging ◽

Controllable Factors ◽

Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157103 ◽

1989 ◽

Vol 47 ◽

pp. 1024-1025

Author(s):

George C. Ruben ◽

William Krakow

Keyword(s):

Single Molecule ◽

Helical Structure ◽

Optical Diffraction ◽

Maximum Diameter ◽

Primary Cell Wall ◽

Cellulose Synthesis ◽

Left Hand ◽

Left Handed ◽

Freeze Dried ◽

Diffraction Patterns

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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