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Abstract(s)
Skeletal muscle is formed by multinucleated myofibers, the biggest cells in the human body. The multiple nuclei in these cells are regularly positioned so that the distance between them is maximized. It was previously found that nuclear positioning is important for skeletal muscle function (Metzger et al., 2012). However, mechanistic insight was missing since no evident structural abnormalities were found as a consequence of nuclear mispositioning. We hypothesized that each nucleus influences the nearby cytoplasm by determining mRNA localization along myofibers. As a consequence, protein translation and regulation would be hampered in situations of nuclear mispositioning, such as in centronuclear myopathies.
Using highly matured mouse myofibers differentiated in vitro, we found that overall mRNA distribution depends on nuclear position. Using smFISH we observed that during myofiber maturation and myofibril organization, mRNAs are pushed towards the sarcolemma. We also validated the nuclear domain theory (Pavlath et al., 1989) by detecting total mRNA clustering around peripheral nuclei. This seems to be the default localization of mRNAs in myofibers since both muscle specific and housekeeping transcripts display the same pattern.
This perinuclear clustering is an active mechanism, dependent on the minus end directed microtubule motor dynein and its activator dynactin. We have also established that the levels of protein translation can depend on nuclear location. Ribosome content is higher in the nuclear region, independently of Dynactin2 expression. Using a heterokaryon system, we show that at least some proteins in the cell remain localized close to their nucleus of origin. Moreover, contractibility of the cells correlates with the position of the nucleus and thus with overall mRNA localization.
Interestingly, a peculiar subset of mRNAs localizes regardless of where the nucleus is placed. A common feature of these transcripts is their extremely big length. We confirmed that this differential distribution is also happening in vivo. We propose that an active mechanism is responsible for this “giant” mRNA localization in order to ensure and facilitate the localization of the encoded proteins. Understanding the mechanisms of mRNA transport and anchoring that govern its subcellular destinations in myofibers may be the key to understand how nuclear positioning impacts muscle activity.
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Keywords
Skeletal muscle mRNA localization Microtubules Translation Contraction Teses de doutoramento - 2019