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

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.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts

“…44 A similar strand-specific telomere-repeat containing non-coding RNAs were detected on the lampbrush chromosomes of turkey (Meleagris gallopavo) and domestic pigeon (Columba livia) 44 and subsequently Japanese quail (Coturnix japonica). 45 In each case the labeled RNP-matrix was revealed only after hybridization with (TAACCC) 5 probe; no labeling was observed in experiments with (TTAGGG) 5 probe. 44 Thus, it was demonstrated that C-rich strand serves as a template for transcription of the avian telomere repeat.…”

“…50 Likewise the telomere repeat transcripts on giant loops-containing termini of chicken and pigeon lampbrush chromosomes can be associated with RNP-rich structures, called GITERA (GIant TErminal RNP Aggregates). 45 However, transcription of the telomere repeat on its own does not induce GITERA formation. GITERA probably serves as nuclear domains for accumulation or nuclear retention of non-nascent poly(A) RNAs, including telomere repeat transcripts, subtelomere RNAs and specific sets of RNAbinding proteins.…”

“…GITERA probably serves as nuclear domains for accumulation or nuclear retention of non-nascent poly(A) RNAs, including telomere repeat transcripts, subtelomere RNAs and specific sets of RNAbinding proteins. 45 Evolutionary conserved pattern of telomere (TTAGGG)n repeat containing RNA transcription is an illustration of the common denominator of genome expression among Vertebrata.…”

“…56 The RNPmatrix of lateral loops carrying telomere RNA is enriched with splicing factor PSF/SFPQ. 45 Thus co-transcriptional stages of satellite RNA processing differ from co-transcriptional stages of mRNA processing.…”

“…84,85 It should be noted that it is quite difficult to obtain mammalian female gametes preparations, especially preparations of oocytes at the first meiotic division, which limited the studies of the biological role of TERRA RNA and TERT in gametogenesis. Transcription of telomere repeat that was demonstrated at the termini of lampbrush chromosomes 40,44,45 makes amphibian and avian oocytes an attractive model for elucidation of TERRA RNA functions and biogenesis.…”