Limits...
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.

Show MeSH

Related in: MedlinePlus

PUF-8 colocalises with the nuclear cap-binding protein NCBP-2. The expression pattern of NCBP-2 as revealed by immunostaining with anti-human CBP20 antibody, the human orthologue of NCBP-2, is shown top left. All other images show the expression patterns of the indicated transgene-expressed fusion proteins. In the bottom row, single nuclei from each panel are shown at higher magnification to the right. Scale bars: 10 μm (top and bottom left); 5 μm (bottom right).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585663&req=5

Figure 5: PUF-8 colocalises with the nuclear cap-binding protein NCBP-2. The expression pattern of NCBP-2 as revealed by immunostaining with anti-human CBP20 antibody, the human orthologue of NCBP-2, is shown top left. All other images show the expression patterns of the indicated transgene-expressed fusion proteins. In the bottom row, single nuclei from each panel are shown at higher magnification to the right. Scale bars: 10 μm (top and bottom left); 5 μm (bottom right).

Mentions: If NCBP-2 indeed interacts with PUF-8, then these proteins would colocalise. To test this, we generated double-transgenic lines expressing both cMyc::mCherry::NCBP-2 and PUF-8::GFP fusion proteins. As shown in Fig. 5, these proteins exhibited significant colocalisation in the nucleus. Unfortunately, owing to the low levels of PUF-8::GFP, we were unable to detect it in western blots and, as a consequence, we could not test the interaction between NCBP-2 and PUF-8 using the co-immunoprecipitation strategy.


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 colocalises with the nuclear cap-binding protein NCBP-2. The expression pattern of NCBP-2 as revealed by immunostaining with anti-human CBP20 antibody, the human orthologue of NCBP-2, is shown top left. All other images show the expression patterns of the indicated transgene-expressed fusion proteins. In the bottom row, single nuclei from each panel are shown at higher magnification to the right. Scale bars: 10 μm (top and bottom left); 5 μm (bottom right).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585663&req=5

Figure 5: PUF-8 colocalises with the nuclear cap-binding protein NCBP-2. The expression pattern of NCBP-2 as revealed by immunostaining with anti-human CBP20 antibody, the human orthologue of NCBP-2, is shown top left. All other images show the expression patterns of the indicated transgene-expressed fusion proteins. In the bottom row, single nuclei from each panel are shown at higher magnification to the right. Scale bars: 10 μm (top and bottom left); 5 μm (bottom right).
Mentions: If NCBP-2 indeed interacts with PUF-8, then these proteins would colocalise. To test this, we generated double-transgenic lines expressing both cMyc::mCherry::NCBP-2 and PUF-8::GFP fusion proteins. As shown in Fig. 5, these proteins exhibited significant colocalisation in the nucleus. Unfortunately, owing to the low levels of PUF-8::GFP, we were unable to detect it in western blots and, as a consequence, we could not test the interaction between NCBP-2 and PUF-8 using the co-immunoprecipitation strategy.

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