STUDIES OF mRNA TRANSPORT AND LOCALIZATION

During mRNA biogenesis, a subset of newly exported mRNA is assembled into larger RNA granules which are transported to subcellular compartments for localized translation. These mechanisms are critical for asymmetric mRNA and protein distribution and have profound impact on cellular physiology. Yet, they are not fully understood at the molecular level. Transport and localization require cis-acting elements on the RNA that are recognized by cellular transacting factors. The main objective of this thesis has been to elucidate the mechanisms that lead to active mRNA sorting to specialized subcellular compartments for translation. Cytoplasmic transport and localization of certain mRNAs is mediated by a well characterized cis-acting element termed RNA trafficking sequence (RTS) found in the untranslated regions (UTR). In paper 1, we discovered that the heterogeneous nuclear ribonucleoprotein CBF-A binds the RTS in the 3’ UTR of the myelin basic protein (MBP) mRNA. CBF-A binding to the RTS occurs both in the nucleus and in the cytoplasm of oligodendrocytes. Since CBF-A gene knockdown impairs MBP mRNA transport to the myelin compartment, we conclude that CBF-A is a novel transacting factor that mediates MBP mRNA transport and localization via the RTS pathway. In neurons mRNA is present in dendrites and synapses but the mechanisms that mediate transport of specific transcripts are not understood in detail. In paper 2, we show that in hippocampal neurons CBF-A functions as transacting factor for transport of Arc, CaMKIIα and BDNF mRNAs to dendrites. This mechanism is RTS-mediated since CBF-A binds to RTS-like sequences in the UTRs of the transcripts and it is dependent on postsynaptic receptor activation. In the nucleus of brain cells CBF-A is excluded from dense chromatin and localizes to nascent pre-mRNPs in perichromatin region. Based on the the fact that CBF-A specifically interact with transcripts that contain RTS sequences, we propose that co-transcriptional RTS-binding by CBF-A may provide de facto a sorting mechanism for transport-competent neuronal mRNAs at an early stage in RNP biogenesis. In spermatogenic cells expression of haploid mRNA is temporally and spatially regulated, partly by controlling mRNA trafficking. In paper 3 we report that CBF-A is involved in transport and localization of protamine 2 mRNA during spermatogenesis. CBF-A binds to the conserved RTS in the protamine 2 mRNA 3’ UTR and in round spermatids, CBF-A accompanies protamine 2 mRNA to chromatoid body. The larger p42 CBF-A splice variant also appears in protamine 2 mRNA-containing polysomes and interacts with the 5’ mRNA cap structure. Since the smaller p37 isoform is excluded from polysomes, we propose that both CBF-A splice variants associate with protamine 2 mRNA and together transit through chromatoid body. In elongating spermatids in response to developmental cue calls when a distinct protamine 2 mRNP emerges in the cytoplasm to engage the translation machinery, the larger p42 isoform remains associated with the transcript via the RTS, presumably to facilitate targeting to the translation machinery. LIST OF PUBLICATIONS I. Chandrasekhar S. Raju, Christian Goritz, Ylva Nord, Ola Hermanson, Carmen Lopez-Iglesias, Neus Visa, Goncalo Castelo-Branco and Piergiorgio Percipalle. In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences Molecular Biology of the Cell, 2008, Vol. 19, 3008–3019 II. Chandrasekhar S. Raju, Nanaho Fukuda, Carmen Lopez-Iglesias, Christian Goritz, Neus Visa and Piergiorgio Percipalle. In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements Molecular Biology of the Cell ,2011, in press; doi:10.1091/E10-11-0904, III. Nanaho Fukuda, Chandrasekhar S. Raju, Tomoyuki Fukuda and Piergiorgio Percipalle. In mouse testis protamine 2 mRNA transport and localized translation is regulated by the transacting factor CBF-A via the A2RE/RTS pathway during spermatogenesis Manuscript Publication not included in this thesis I. Piergiorgio Percipalle, Chandrasekhar S. Raju and Nanaho Fukuda. Actin-associated hnRNP proteins as transacting factors in the control of mRNA transport and localization. RNA biology, 2009, 6; 171 174