Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

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Control of noncoding RNA production and histone levels by a 5′ tRNA fragment

10.1101/498949 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ana Boskovic ◽

Xin Yang Bing ◽

Ebru Kaymak ◽

Oliver J Rando

Keyword(s):

Transcriptional Repression ◽

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Noncoding Rnas ◽

Cajal Body ◽

Mrna Levels ◽

Histone 3 ◽

Rna Biogenesis ◽

Mechanistic Basis

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs", are an emerging class of regulatory RNAs with poorly understood functions in cellular regulation. We recently identified a role for one specific tRF - 5′ tRF-Gly-GCC, or tRF-GG - in repression of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs normally synthesized in Cajal bodies. Among these noncoding RNAs, tRF-GG regulation of the U7 snRNA modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, activity of a histone 3′ UTR reporter, and ultimately on MERVL regulation could all be suppressed by the U7 RNA. We show that the related RNA-binding proteins hnRNPF and H bind directly to tRF-GG, and are required for Cajal body biogenesis. Together, our data reveal a conserved mechanism for 5′ tRNA fragment control of noncoding RNA biogenesis and, consequently, in global chromatin organization.

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Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

10.1101/454066 ◽

2018 ◽

Cited By ~ 7

Author(s):

Alejandro Padròn ◽

Shintaro Iwasaki ◽

Nicholas T. Ingolia

Keyword(s):

Translation Initiation ◽

Messenger Rna ◽

Spatial Organization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Granules ◽

Ribonucleoprotein Complexes ◽

Spatial Environment ◽

Dynamic Structures ◽

As Stress

AbstractDiverse ribonucleoprotein complexes control messenger RNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-Seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS) provides new insights into the organization of translation initiation complexes on active mRNAs, as well as exposing unanticipated complexity in stress granule composition, and provides a powerful and general approach to explore the spatial environment of macromolecules.

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