A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation

Nader Ezzeddine, Jiandong Chen, Bernhard Waltenspiel, Brandon Burch, Todd Albrecht, Ming Zhuo, William D. Warren, William F. Marzluff, Eric Wagner

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3′-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3′-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3′-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3′-end formation and found that processing was strongly dependent upon nucleotides located within the 3′ stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3′ box. Substitution of the actin promoter for the snRNA promoter abolished proper 3′-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3′-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3′-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.

Original languageEnglish (US)
Pages (from-to)328-341
Number of pages14
JournalMolecular and Cellular Biology
Volume31
Issue number2
DOIs
StatePublished - Jan 2011
Externally publishedYes

Fingerprint

Small Nuclear RNA
Drosophila Proteins
Drosophila
Vertebrates
mRNA Cleavage and Polyadenylation Factors
RNA Polymerase II
Uridine
RNA Precursors
Green Fluorescent Proteins
Drosophila melanogaster
Diptera
Histones
U7 small nuclear RNA
Actins
Nucleotides
Gene Expression
Messenger RNA
Mutation
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation. / Ezzeddine, Nader; Chen, Jiandong; Waltenspiel, Bernhard; Burch, Brandon; Albrecht, Todd; Zhuo, Ming; Warren, William D.; Marzluff, William F.; Wagner, Eric.

In: Molecular and Cellular Biology, Vol. 31, No. 2, 01.2011, p. 328-341.

Research output: Contribution to journalArticle

Ezzeddine, N, Chen, J, Waltenspiel, B, Burch, B, Albrecht, T, Zhuo, M, Warren, WD, Marzluff, WF & Wagner, E 2011, 'A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation', Molecular and Cellular Biology, vol. 31, no. 2, pp. 328-341. https://doi.org/10.1128/MCB.00943-10
Ezzeddine, Nader ; Chen, Jiandong ; Waltenspiel, Bernhard ; Burch, Brandon ; Albrecht, Todd ; Zhuo, Ming ; Warren, William D. ; Marzluff, William F. ; Wagner, Eric. / A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation. In: Molecular and Cellular Biology. 2011 ; Vol. 31, No. 2. pp. 328-341.
@article{f213375a4b854ae88457b494b835d108,
title = "A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation",
abstract = "Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3′-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3′-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3′-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3′-end formation and found that processing was strongly dependent upon nucleotides located within the 3′ stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3′ box. Substitution of the actin promoter for the snRNA promoter abolished proper 3′-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3′-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3′-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.",
author = "Nader Ezzeddine and Jiandong Chen and Bernhard Waltenspiel and Brandon Burch and Todd Albrecht and Ming Zhuo and Warren, {William D.} and Marzluff, {William F.} and Eric Wagner",
year = "2011",
month = "1",
doi = "10.1128/MCB.00943-10",
language = "English (US)",
volume = "31",
pages = "328--341",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "2",

}

TY - JOUR

T1 - A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3′-end formation

AU - Ezzeddine, Nader

AU - Chen, Jiandong

AU - Waltenspiel, Bernhard

AU - Burch, Brandon

AU - Albrecht, Todd

AU - Zhuo, Ming

AU - Warren, William D.

AU - Marzluff, William F.

AU - Wagner, Eric

PY - 2011/1

Y1 - 2011/1

N2 - Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3′-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3′-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3′-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3′-end formation and found that processing was strongly dependent upon nucleotides located within the 3′ stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3′ box. Substitution of the actin promoter for the snRNA promoter abolished proper 3′-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3′-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3′-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.

AB - Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3′-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3′-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3′-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3′-end formation and found that processing was strongly dependent upon nucleotides located within the 3′ stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3′ box. Substitution of the actin promoter for the snRNA promoter abolished proper 3′-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3′-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3′-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.

UR - http://www.scopus.com/inward/record.url?scp=78751472594&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78751472594&partnerID=8YFLogxK

U2 - 10.1128/MCB.00943-10

DO - 10.1128/MCB.00943-10

M3 - Article

C2 - 21078872

AN - SCOPUS:78751472594

VL - 31

SP - 328

EP - 341

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 2

ER -