Chemistry Reference
In-Depth Information
Another important characteristic of RNA molecules is that they are never naked
in the cell. They are always associated with proteins, forming ribonucleoprotein
particles (RNPs). As a consequence, the RNP is much higher in molecular weight
than the RNA alone. Moreover, the composition of the complex is modi
ed over time
due to exchange of binding partners, therefore increasing the complexity and the
spectrumof possible interactions and functions. These characteristicsmake the RNA
molecule, in all its forms and functions, an exciting and important object of study.
Many questions have been raised about RNP dynamics in the nucleus and in the
cytoplasm. Some of them concern how RNAs move, whether they follow rules of free
diffusion or energy-dependent movement, and to what extent the environment, such
as chromatin in the nucleus or
filament networks in the cytoplasm, constrains RNA
movements. RNA dynamics range from the sites of nuclear transcription, where
maturation occurs, to the speci
c localization of particular RNAs in the cytoplasm,
which creates de
ned gradients by enrichment in, or exclusion from, particular areas.
8.2
RNA Visualization inside Cells
Because seeing is believing, during the last decade efforts have been focused on
observing the actual dynamics of RNA movement inside a single living cell.
In the next section we outline the most important components to be considered
when imaging mRNAs during their movements in living cells: the development of
suitable methods to label the RNA, generating a suf
cient signal to detect speci
c
individual transcripts, and improvements in imaging technologies. In the subse-
quent sections we will describe the travel of RNA molecules from transcription sites
until their
final destination in their respective translational compartments.
8.2.1
Techniques to Label RNA
The intrinsic complexity of the cellular system gives rise to many issues. To address
them, different methods have been developed to visualize RNAs. Before choosing a
particular approach, pros and cons have to be considered taking into account the
cellular system, the target and the aim of the project.
One among the
first techniques utilized to study the RNA dynamics in living cells
was Fluorescent In Vivo Hybridization (FIVH, [3]). This method was developed on
the basis of the Fluorescent In SituHybridization (FISH, [4
6]) and relies on intrinsic
abilities of oligonucleotides to recognize and hybridize to a complementary target
sequence. The main difference between the two techniques is that FISH applies to
fixed cells, while FIVH allows the study of transcripts in living cells. The
rst
methodological improvement of the FIVH technique was the optimization of
protocols for oligonucleotide uptake and hybridization in vivo. Fluorescent [3] or
caged-
uorescent oligo-dT [7] were used to probe the poly(A) tails of all mRNAs and
study their movements. The ability to obscure the
uorochrome on the probe by a
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