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Giant poly(A)-rich RNP aggregates form at terminal regions of avian lampbrush chromosomes

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Abstract

The cell nucleus comprises a number of chromatin-associated domains. Certain chromatin-associated domains are nucleated by nascent RNA and accumulate non-nascent transcripts in the form of ribonucleoprotein (RNP) aggregates. In the transcriptionally active nucleus of the growing avian oocyte, RNP-rich structures, here termed giant terminal RNP aggregates (GITERA), form at the termini of lampbrush chromosomes. Using GITERA as an example, we aimed to explore mechanisms of RNP aggregate formation at certain chromosomal loci to establish whether they accumulate non-nascent RNA and to analyze protein composition in RNP aggregates. We found that GITERA on chicken and pigeon lampbrush chromosomes do not contain nascent transcripts. At the same time, RNA fluorescent in situ hybridization (FISH) and in situ reverse transcription demonstrated that GITERA accumulate poly(A)-rich RNA. Moreover, subtelomere chromosome regions adjacent to GITERA are transcriptionally active as shown by detection of incorporated BrUTP and the elongating form of RNA polymerase II. GITERA on both chicken and pigeon lampbrush chromosomes are enriched in splicing factors but not in heterogeneous nuclear RNP (hnRNP) L and K. A subtype of GITERA concentrates hnRNP I/PTB and p54nrb/NonO. Interestingly, hnRNP I/PTB and p54nrb/NonO in such subtype of GITERA were revealed in long threads. The resemblance of these threads to amyloid-like fibers is discussed. Our data suggest that transcription of subtelomeric sequences serves as a seeding event for accumulation of non-nascent RNA and associated RNP proteins. Such accumulation leads to GITERA formation in terminal chromosomal regions in avian oocyte nucleus. 3′-processed transcripts derived from other chromosomal loci may be attracted to GITERA by binding to the same RNP proteins or to their interaction partners.

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Abbreviations

FISH:

Fluorescent in situ hybridization

FUS:

Fused in sarcoma

GITERA:

Giant terminal RNP aggregates

hnRNP:

Heterogeneous nuclear RNP

ISRT:

In situ reverse transcription

mAb:

Monoclonal antibody

PSF/SFPQ:

PTB-associated splicing factor/splicing factor proline- and glutamine-rich

p54nrb/NonO:

nuclear RNA-binding protein/non-POU domain-containing octamer-binding protein

PTB:

Polypyrimidine tract-binding protein

RNase A:

Ribonuclease A

SC35:

Splicing component (factor) 35 kDa

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Acknowledgments

We are grateful to Ulrich Scheer (University of Wuerzburg, Germany) for kindly providing mAb V22. We also would like to thank Svetlana Deryusheva (Carnegie Institution for Science, USA) for helpful methodological advises. RNP composition analysis was supported by the Russian Science Foundation (grant #14-14-00131); poly(A)-RNA distribution analysis was supported by the grant of the President of Russia (grant #NS-3553.2014.4). The work was partially performed using experimental equipment of the Research Resource Centers “Chromas” and “Molecular and Cell Technologies” of St. Petersburg State University.

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Kulikova, T., Chervyakova, D., Zlotina, A. et al. Giant poly(A)-rich RNP aggregates form at terminal regions of avian lampbrush chromosomes. Chromosoma 125, 709–724 (2016). https://doi.org/10.1007/s00412-015-0563-4

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