Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

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Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes. / Rugjee, Kushal Nivriti; Roy Chaudhury, Subhendu; Al-jubran, Khalid; Ramanathan, Preethi; Matina, Annunziata; Wen, Jikai; Brogna, Saverio.

In: PeerJ, Vol. 1, e15, 12.02.2013.

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Rugjee, Kushal Nivriti ; Roy Chaudhury, Subhendu ; Al-jubran, Khalid ; Ramanathan, Preethi ; Matina, Annunziata ; Wen, Jikai ; Brogna, Saverio. / Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes. In: PeerJ. 2013 ; Vol. 1.

Bibtex

@article{f7d59884c0f54e57ac7b0e09e4e16aa0,
title = "Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes",
abstract = "Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs.",
keywords = "Ribosomal proteins, Drosophila, Polytene chromosomes, Visualization",
author = "Rugjee, {Kushal Nivriti} and {Roy Chaudhury}, Subhendu and Khalid Al-jubran and Preethi Ramanathan and Annunziata Matina and Jikai Wen and Saverio Brogna",
year = "2013",
month = feb,
day = "12",
doi = "10.7717/peerj.15",
language = "English",
volume = "1",
journal = "PeerJ",
issn = "2167-8359",
publisher = "PeerJ",

}

RIS

TY - JOUR

T1 - Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

AU - Rugjee, Kushal Nivriti

AU - Roy Chaudhury, Subhendu

AU - Al-jubran, Khalid

AU - Ramanathan, Preethi

AU - Matina, Annunziata

AU - Wen, Jikai

AU - Brogna, Saverio

PY - 2013/2/12

Y1 - 2013/2/12

N2 - Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs.

AB - Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs.

KW - Ribosomal proteins

KW - Drosophila

KW - Polytene chromosomes

KW - Visualization

U2 - 10.7717/peerj.15

DO - 10.7717/peerj.15

M3 - Article

C2 - 23638349

VL - 1

JO - PeerJ

JF - PeerJ

SN - 2167-8359

M1 - e15

ER -