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PUF-8 and TCER-1 are essential for normal levels of multiple mRNAs in the C. elegans germline.

Pushpa K, Kumar GA, Subramaniam K - Development (2013)

Bottom Line: The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type.These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance.We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.

ABSTRACT
PUF family proteins are well-conserved regulators of cell proliferation in different developmental processes. They regulate target mRNAs by promoting degradation or by influencing translation through interaction with the translation initiation machinery. Here we show that Caenorhabditis elegans PUF-8 functions redundantly with the nuclear protein TCER-1 in the post-transcriptional maintenance of at least six germline mRNAs. The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type. These two proteins colocalise at the inner nuclear periphery, and their absence leads to reduced germ cell proliferation and to sterility. A yeast two-hybrid screen of 31 components of the nuclear pore complex and mRNA processing machineries identified seven proteins involved in mRNA export as potential partners of PUF-8. One of these, the nuclear cap-binding protein NCBP-2, colocalises with PUF-8 in the nucleus. A 50 amino acid N-terminal domain of PUF-8 is essential for interaction with NCBP-2 and for PUF-8 to function redundantly with TCER-1. These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance. We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.

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PUF-8 and TCER-1 colocalise at the inner nuclear periphery. (A) Expression pattern of tcer-1::gfp transgene in a wild-type background (top) and of gfp::lem-2, a nuclear envelope marker, and tcer-1::mCherry transgenes (bottom). Only a few germ cells from the distal part of the gonad are shown at higher magnification. Left, distal; right, proximal. (B) Row 1 (from top) shows expression patterns of PUF-8::GFP and mCherry::EMR-1, another nuclear envelope marker, in worms carrying both transgenes. Row 3, PUF-8 is fused to mCherry and the nuclear envelope is visualised using the GFP::LEM-2 marker. Row 5, expression patterns of GLD-1::GFP (shown here as a control) and mCherry::EMR-1. Rows 2, 4 and 6 are as rows 1, 3 and 5, respectively, but after treatment with leptomycin B (LMB). (C) Expression patterns of PUF-8::GFP and TCER-1::mCherry in worms carrying both transgenes. Beneath, two nuclei from each panel shown at a higher magnification. Except for A (top), images have been deconvolved using the Iterative Deconvolution module of Axiovision software to enhance the axial resolution of the fluorescence signal. (B,C) Only a part of the distal gonad, revealing a few germ cell nuclei, is shown. Scale bars: 25 μm in A (top); 10 μm in B and C (top); 5 μm in A (bottom) and C (bottom).
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Figure 2: PUF-8 and TCER-1 colocalise at the inner nuclear periphery. (A) Expression pattern of tcer-1::gfp transgene in a wild-type background (top) and of gfp::lem-2, a nuclear envelope marker, and tcer-1::mCherry transgenes (bottom). Only a few germ cells from the distal part of the gonad are shown at higher magnification. Left, distal; right, proximal. (B) Row 1 (from top) shows expression patterns of PUF-8::GFP and mCherry::EMR-1, another nuclear envelope marker, in worms carrying both transgenes. Row 3, PUF-8 is fused to mCherry and the nuclear envelope is visualised using the GFP::LEM-2 marker. Row 5, expression patterns of GLD-1::GFP (shown here as a control) and mCherry::EMR-1. Rows 2, 4 and 6 are as rows 1, 3 and 5, respectively, but after treatment with leptomycin B (LMB). (C) Expression patterns of PUF-8::GFP and TCER-1::mCherry in worms carrying both transgenes. Beneath, two nuclei from each panel shown at a higher magnification. Except for A (top), images have been deconvolved using the Iterative Deconvolution module of Axiovision software to enhance the axial resolution of the fluorescence signal. (B,C) Only a part of the distal gonad, revealing a few germ cell nuclei, is shown. Scale bars: 25 μm in A (top); 10 μm in B and C (top); 5 μm in A (bottom) and C (bottom).

Mentions: TCER-1 has been annotated as a transcription elongation regulator based on its similarity to the mammalian protein CA150 (TCERG1) and its yeast orthologue PRP40p (Ghazi et al., 2009). CA150 has been shown to be essential for mRNA processing and, based on its ability to interact with the C-terminal domain (CTD) of RNA polymerase II and the components of the spliceosome, CA150 has been implicated in coupling transcription and splicing (Lin et al., 2004; Pearson et al., 2008). To begin to address its functional redundancy with PUF-8, we determined the distribution pattern of TCER-1 in the germline using TCER-1::GFP and TCER-1::mCherry transgenes. Both of these transgenes were expressed using the promoter and downstream sequences of tcer-1, and were able to restore fertility in puf-8(-) tcer-1(-) worms. As shown in Fig. 2A, TCER-1::GFP was present in all germ cell nuclei, starting from the mitotic cells at the distal end to the oocytes at the proximal end of the gonad. TCER-1::mCherry showed an identical expression pattern (data not shown). Both of these transgenes were expressed in somatic nuclei as well (data not shown).


PUF-8 and TCER-1 are essential for normal levels of multiple mRNAs in the C. elegans germline.

Pushpa K, Kumar GA, Subramaniam K - Development (2013)

PUF-8 and TCER-1 colocalise at the inner nuclear periphery. (A) Expression pattern of tcer-1::gfp transgene in a wild-type background (top) and of gfp::lem-2, a nuclear envelope marker, and tcer-1::mCherry transgenes (bottom). Only a few germ cells from the distal part of the gonad are shown at higher magnification. Left, distal; right, proximal. (B) Row 1 (from top) shows expression patterns of PUF-8::GFP and mCherry::EMR-1, another nuclear envelope marker, in worms carrying both transgenes. Row 3, PUF-8 is fused to mCherry and the nuclear envelope is visualised using the GFP::LEM-2 marker. Row 5, expression patterns of GLD-1::GFP (shown here as a control) and mCherry::EMR-1. Rows 2, 4 and 6 are as rows 1, 3 and 5, respectively, but after treatment with leptomycin B (LMB). (C) Expression patterns of PUF-8::GFP and TCER-1::mCherry in worms carrying both transgenes. Beneath, two nuclei from each panel shown at a higher magnification. Except for A (top), images have been deconvolved using the Iterative Deconvolution module of Axiovision software to enhance the axial resolution of the fluorescence signal. (B,C) Only a part of the distal gonad, revealing a few germ cell nuclei, is shown. Scale bars: 25 μm in A (top); 10 μm in B and C (top); 5 μm in A (bottom) and C (bottom).
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Figure 2: PUF-8 and TCER-1 colocalise at the inner nuclear periphery. (A) Expression pattern of tcer-1::gfp transgene in a wild-type background (top) and of gfp::lem-2, a nuclear envelope marker, and tcer-1::mCherry transgenes (bottom). Only a few germ cells from the distal part of the gonad are shown at higher magnification. Left, distal; right, proximal. (B) Row 1 (from top) shows expression patterns of PUF-8::GFP and mCherry::EMR-1, another nuclear envelope marker, in worms carrying both transgenes. Row 3, PUF-8 is fused to mCherry and the nuclear envelope is visualised using the GFP::LEM-2 marker. Row 5, expression patterns of GLD-1::GFP (shown here as a control) and mCherry::EMR-1. Rows 2, 4 and 6 are as rows 1, 3 and 5, respectively, but after treatment with leptomycin B (LMB). (C) Expression patterns of PUF-8::GFP and TCER-1::mCherry in worms carrying both transgenes. Beneath, two nuclei from each panel shown at a higher magnification. Except for A (top), images have been deconvolved using the Iterative Deconvolution module of Axiovision software to enhance the axial resolution of the fluorescence signal. (B,C) Only a part of the distal gonad, revealing a few germ cell nuclei, is shown. Scale bars: 25 μm in A (top); 10 μm in B and C (top); 5 μm in A (bottom) and C (bottom).
Mentions: TCER-1 has been annotated as a transcription elongation regulator based on its similarity to the mammalian protein CA150 (TCERG1) and its yeast orthologue PRP40p (Ghazi et al., 2009). CA150 has been shown to be essential for mRNA processing and, based on its ability to interact with the C-terminal domain (CTD) of RNA polymerase II and the components of the spliceosome, CA150 has been implicated in coupling transcription and splicing (Lin et al., 2004; Pearson et al., 2008). To begin to address its functional redundancy with PUF-8, we determined the distribution pattern of TCER-1 in the germline using TCER-1::GFP and TCER-1::mCherry transgenes. Both of these transgenes were expressed using the promoter and downstream sequences of tcer-1, and were able to restore fertility in puf-8(-) tcer-1(-) worms. As shown in Fig. 2A, TCER-1::GFP was present in all germ cell nuclei, starting from the mitotic cells at the distal end to the oocytes at the proximal end of the gonad. TCER-1::mCherry showed an identical expression pattern (data not shown). Both of these transgenes were expressed in somatic nuclei as well (data not shown).

Bottom Line: The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type.These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance.We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.

ABSTRACT
PUF family proteins are well-conserved regulators of cell proliferation in different developmental processes. They regulate target mRNAs by promoting degradation or by influencing translation through interaction with the translation initiation machinery. Here we show that Caenorhabditis elegans PUF-8 functions redundantly with the nuclear protein TCER-1 in the post-transcriptional maintenance of at least six germline mRNAs. The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type. These two proteins colocalise at the inner nuclear periphery, and their absence leads to reduced germ cell proliferation and to sterility. A yeast two-hybrid screen of 31 components of the nuclear pore complex and mRNA processing machineries identified seven proteins involved in mRNA export as potential partners of PUF-8. One of these, the nuclear cap-binding protein NCBP-2, colocalises with PUF-8 in the nucleus. A 50 amino acid N-terminal domain of PUF-8 is essential for interaction with NCBP-2 and for PUF-8 to function redundantly with TCER-1. These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance. We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.

Show MeSH
Related in: MedlinePlus