User:Gonzalezmg1/sandbox

Three-Dimensional Organization of Polyribosomes- A Modern Approach


 * good information about the different structures of polyribosomes and polyribosome configuration
 * development of cryo electron tomography
 * confirmation of previous observations regarding structure
 * discusses the participation of mRNA in transport and localization of protein synthesis

Translatome profiling: methods for genome-scaling analysis of mRNA translation.


 * discusses the use of polysomal profiling to analyze the distribution of specific mRNAs
 * profiling is used to compare the translational status of mRNAs in different cell types
 * compares 3 different methods of profiling: listing advantages and disadvantages

Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis

Rough Draft of Polysome Article
 * all miRNAs are associated with membrane bound polysomes, as opposed to polysomes in general
 * discusses protocol for isolation of free and membrane-bound polysomes

**** NOTE: references were not able to be verified, entire article had to be rewritten

Polysome
(unedited text: 3/2/20)

A polyribosome (or polysome or ergasome) is a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides. Originally coined "ergosomes" in 1963, they were further characterized by Jonathan Warner, Paul M. Knopf, and Alex Rich. The simultaneous analysis of the polysome with all the peptides it produces under given conditions (the translatome) leads to a better insight of the complexity of how mutations, extracellular stimuli, intercellular cues, growth conditions, and stress could lead to a change in translation in the cell. Polysomes consist of varying numbers of ribosomes and each ribosome contributes to the addition of its substantial mass. They act as a platform for signalling molecules to get involved in the emergence of polypeptides and ribosomes, and aid the nascent polypeptides to fold in the presence of chaperones, which later impact the function of synthesized polysomes.

(edited text: 3/2/20)

A polysome, or polyribosome, is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. Polysomes are formed during the elongation phase when ribosomes and elongation factors synthesize the encoded polypeptide. Multiple ribosomes move along the coding region of mRNA, creating a polysome. The ability of multiple ribosomes to function on an mRNA molecule explains the limited abundance of mRNA in the cell. Polyribosome structure differs between prokaryotic polysomes, eukaryotic polysomes, and membrane bound polysomes. Polysome activity can be used to measure the level of gene expression through a technique called polysomal profiling.

Structure
(unedited: 3/2/20)

Atomic-force microscopy (AFM) was used to obtain the images of polysomes from mouse brain samples in air and liquid. The images were then reconstructed to depict structures in three-dimensions, thereby facilitating the study of polysome organization. Polysomes can be directly visualized by electron microscopy because they form very high molecular weight particles. Many ribosomes simultaneously read one mRNA polypeptide by progressing along the mRNA chain to synthesize the same protein. They may appear as linear polysomes or circular rosettes with microscopy, but are mainly circular in vivo. This circularization is aided by the fact that mRNA is able to be twisted into a circular formation, allowing a cycle of rapid ribosome recycling to occur. The 5' 7-methylguanosine cap and 3' poly(A) tail present on eukaryotic mRNA aid in this process.

(edited: 3/2/20)

Introduction to Structure Determination
Electron microscopy technologies such as staining, metal shadowing, and ultra-thin cell sections were the original methods to determine polysome structure. The development of cryo-electron microscopy techniques has allowed for increased resolution of the image, leading to a more precise method to determine structure. Different structural configurations of polyribosomes could reflect a variety in translation of mRNAs. An investigation of the ratio of polyribosomal shape elucidated that a high number of circular and zigzag polysomes were found after several rounds of translation. A longer period of translation caused the formation of densely packed 3-D helical polysomes. Different cells produce different structures of polysomes.

Prokaryotic Polysomes
Prokaryotic polysomes have been found to form double-row structures. In this conformation, the ribosomes are contacting each other through smaller subunits. These double row structures generally have a “sinusoidal” (zigzag) or 3-D helical path. In the “sinusoidal” path, there are two types of contact between the small subunits- “top-to-top” or “top-to-bottom”. In the 3-D helical path, only “top-to-top” contact is observed.

in situ
in situ (in cell) studies have shown that eukaryotic polysomes exhibit linear configurations. Densely packed 3-D helices and planar double-row polysomes were found with variable packing including “top-to-top” contacts similar to prokaryotic polysomes. Eukaryotic 3-D polyribosomes are similar to prokaryotic 3-D polyribosomes in that they are “densely packed left-handed helices with four ribosomes per turn”. This dense packing can determine their function as regulators of translation, with 3-D polyribosomes being found in sarcoma cells using fluorescence microscopy.

in vitro
Atomic force microscopy used in in vitro studies have shown that circular eukaryotic polysomes can be formed by free polyadenylated mRNA in the presence of initiation factor eIF4E bound to the 5’ cap and PABP bound to the 3’-poly(A) tail. However, this interaction between cap and poly(A)-tail mediated by a protein complex is not a unique way of circularizing polysomal mRNA. It has been found that topologically circular polyribosomes can be successfully formed in the translational system with mRNA with no cap and no poly(A) tail as well as a capped mRNA without a 3’-poly(A) tail.

Membrane-bound Polysomes
Polyribosomes bound to membranes are restricted by a 2 dimensional space given by the membrane surface. The restriction of inter-ribosomal contacts causes a round-shape configuration that arranges ribosomes along the mRNA so that the entry and exit sites form a smooth pathway. Each ribosome is turned relative to the previous one, resembling a planar spiral.

Polysomal Profiling
Polysomal Profiling is a technique that uses cycloheximide to arrest translation and a sucrose gradient to separate the resulting cell extract by centrifugation. Ribosome-associated mRNAs migrate faster than free mRNAs and polysome associated mRNAs migrate faster than ribosome associated mRNAs. Several peaks corresponding to mRNA are revealed by the measurement of total protein across the gradient. The corresponding mRNA is associated with increasing numbers of ribosomes as polysomes. The presence of mRNA across the gradient reveals the translation of the mRNA. Polysomal profiling is optimally applied to cultured cells and tissues to track the translational status of an identified mRNA as well as measure ribosome density. This technique has been used to compare the translational status of mRNAs in different cell types.

For example, polysomal profiling was used in a study to investigate the effect of vesicular stomatitis virus (VSV) in mammalian cells. The data from polysomal profiling showed that host mRNAs are outcompeted by viral mRNAs for polysomes, therefore decreasing the translation of host mRNA and increasing the translation of viral mRNA.