Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization

Inara B. Lazdins, Michael Delannoy, Barbara Sollner-Webb

Research output: Contribution to journalArticle

Abstract

We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5′ region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5′ end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3′ end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions-fibrillar centers, dense fibrillar components, and granular components-for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.

Original languageEnglish (US)
Pages (from-to)481-495
Number of pages15
JournalChromosoma
Volume105
Issue number7-8
DOIs
StatePublished - Jun 1997

Fingerprint

RNA Precursors
In Situ Hybridization
Ribosomal DNA
Cell Nucleolus
Fluorescence In Situ Hybridization
Ribosomes
Microscopy
Electrons
Light
DNA

ASJC Scopus subject areas

  • Genetics

Cite this

Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization. / Lazdins, Inara B.; Delannoy, Michael; Sollner-Webb, Barbara.

In: Chromosoma, Vol. 105, No. 7-8, 06.1997, p. 481-495.

Research output: Contribution to journalArticle

Lazdins, Inara B. ; Delannoy, Michael ; Sollner-Webb, Barbara. / Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization. In: Chromosoma. 1997 ; Vol. 105, No. 7-8. pp. 481-495.
@article{3d1a418c9dd54c64ae479ad4bc327134,
title = "Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization",
abstract = "We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5′ region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5′ end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3′ end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions-fibrillar centers, dense fibrillar components, and granular components-for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.",
author = "Lazdins, {Inara B.} and Michael Delannoy and Barbara Sollner-Webb",
year = "1997",
month = "6",
doi = "10.1007/BF02510485",
language = "English (US)",
volume = "105",
pages = "481--495",
journal = "Chromosoma",
issn = "0009-5915",
publisher = "Springer Science and Business Media Deutschland GmbH",
number = "7-8",

}

TY - JOUR

T1 - Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization

AU - Lazdins, Inara B.

AU - Delannoy, Michael

AU - Sollner-Webb, Barbara

PY - 1997/6

Y1 - 1997/6

N2 - We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5′ region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5′ end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3′ end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions-fibrillar centers, dense fibrillar components, and granular components-for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.

AB - We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5′ region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5′ end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3′ end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions-fibrillar centers, dense fibrillar components, and granular components-for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.

UR - http://www.scopus.com/inward/record.url?scp=0030786573&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030786573&partnerID=8YFLogxK

U2 - 10.1007/BF02510485

DO - 10.1007/BF02510485

M3 - Article

C2 - 9211976

AN - SCOPUS:0030786573

VL - 105

SP - 481

EP - 495

JO - Chromosoma

JF - Chromosoma

SN - 0009-5915

IS - 7-8

ER -