Intranuclear Pre-mRNA Trafficking in an Insect Model System

Kiesler, Eva; Visa, Neus

doi:10.1007/978-3-540-74266-1_5

Cited by 10 publications

(6 citation statements)

References 85 publications

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“…In spite of the fact that the general organization of the polytene nuclei differs from that of diploid nuclei, the basic processes of gene expression are the same in both types of cells (reviewed in refs. 32 and 33). We have used the advantages that polytene nuclei offer for in situ studies of gene expression to analyze the association of profilin with chromatin.…”

Section: Discussionmentioning

confidence: 98%

Profilin is associated with transcriptionally active genes

Söderberg

Hessle²,

Euler³

et al. 2012

Nucleus

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We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.

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Section: Discussionmentioning

confidence: 98%

Profilin is associated with transcriptionally active genes

Söderberg

Hessle²,

Euler³

et al. 2012

Nucleus

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“…In this study, we have investigated, at the single-particle level, the intranuclear trafficking of native mRNPs in living tissue by using a well-established model system for RNA biogenesis (19). A specific endogenous mRNP particle, BR2 mRNP, was labeled in vivo by fluorescent oligonucleotides complementary to a highly repetitive sequence in BR2 RNA.…”

Section: Discussionmentioning

confidence: 99%

“…The BR particles, Ϸ50 nm in diameter, can be recognized in nucleoplasm and during their translocation through the nuclear pore complexes. Approximately a dozen BR-associated RNAbinding proteins have been identified, some of which stay associated with the transcript when transferred from the gene to the polysomes, e.g., hrp36, whereas others leave the transcript in conjunction with the passage through the nuclear pore complex (12). By immunoelectron microscopy of BrUTP-labeled BR particles, it has been possible to follow the dispersion of the BR particles from the transcription sites into the vast interchromosomal regions of the cell nucleus (13).…”

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confidence: 99%

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

Siebrasse

Veith

Dobay

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10-to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.Balbiani ring particles ͉ in vivo labeling ͉ mRNP trafficking ͉ single-molecule fluorescence microscopy ͉ single-particle tracking C oncomitant with transcription, the growing premessenger RNA (pre-mRNA) molecules become associated with proteins to form ribonucleoprotein (RNP) particles (1). The pre-mRNA is processed to mRNA, and the reorganized messenger RNP (mRNP) particles are prepared for export (2, 3). After being released from the gene, mRNP particles move randomly within the nucleus, apparently by free diffusion (4). However, the mobility can be affected by energy depletion, which suggests that ATP-dependent processes also play a role (5). Recently, it became feasible to track individual mRNP particles in the nucleoplasm (6, 7). It could then be confirmed that the particles move by free diffusion, but the diffusion is often spatially constrained. mRNPs travel in interchromatin channels (8, 9), and the movement of mRNPs is probably hindered in tight channels and even stalled in cavities formed by chromatin. In the single-particle-tracking experiments, ATP depletion showed no effect on the individual, still-mobile mRNPs but further constrained the movement of the particles, probably because of a more condensed organization of the chromatin (6, 7). To further study the movement of mRNPs, we have now chosen an experimental system that allows us to follow the movement of individual mRNPs in large nucleoplasmic regions lacking chromatin, and thus we avoid the complex influence of chromatin. Surprisingly, we then find that the mRNP particles move in a discontinuous fashion, suggesting that the particles transiently interact with supramolecular assemblie...

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“…The Balbiani ring (BR) genes of C. tentans code for large secretory proteins in the salivary glands of the Chironomus larvae (for review, see Wieslander 1994). The BR pre-mRNAs have all the features of protein-coding transcripts and undergo typical premRNA processing (for review, see Wieslander et al 1996;Kiesler and Visa 2004). The BR genes can be identified in polytene chromosome preparations, and by using immunoelectron microscopy (immuno-EM) it is possible to study the association of specific proteins with the newly synthesized BR pre-mRNAs at the site of transcription and on the way to the nuclear pores.…”

Section: Introductionmentioning

confidence: 99%

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

Hessle

Euler²,

Valdivia

et al. 2012

RNA

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Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

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Intranuclear Pre-mRNA Trafficking in an Insect Model System

Cited by 10 publications

References 85 publications

Profilin is associated with transcriptionally active genes

Profilin is associated with transcriptionally active genes

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

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