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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 functions redundantly with tcer-1. Dissected C. elegans adult gonads of the indicated genotype stained with DAPI. Left, distal; right, proximal. The three images are at identical magnification. The double-mutant gonad is significantly smaller than either single mutant and it contains fewer germ cells and no gametes. Scale bars: 25 μm.
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Figure 1: puf-8 functions redundantly with tcer-1. Dissected C. elegans adult gonads of the indicated genotype stained with DAPI. Left, distal; right, proximal. The three images are at identical magnification. The double-mutant gonad is significantly smaller than either single mutant and it contains fewer germ cells and no gametes. Scale bars: 25 μm.

Mentions: To identify gene(s) that potentially compensate for the lack of PUF-8 at 20°C, a synthetic genetic screen was carried out on the puf-8(+/-) background (Vaid et al., 2013). To map one of the synthetic mutants identified in that screen, we performed an RNAi screen of genes present in a limited genetic interval on chromosome II on the puf-8(zh17) genetic background, which is a strong loss-of-function allele. This RNAi screen identified tcer-1 as functionally redundant with puf-8. Consistent with the earlier results, 100% of the puf-8(zh17) worms were fertile at 20°C (n=150). Similarly, all of the tcer-1(RNAi) worms were fertile and did not show any observable defects (n=500). By contrast, the puf-8(zh17) tcer-1(RNAi) double-mutant worms were 100% sterile at 20°C (n=500). Subsequently, we confirmed the RNAi results by generating a puf-8(-) tcer-1(-) genetic double-mutant strain using tcer-1(tm1452), which has a 392 bp deletion that includes most of the second exon of tcer-1. Similar to the tcer-1(RNAi) worms, tcer-1(tm1452) worms were normal (n=500). In the double-mutant worms, the total number of germ cells was severely reduced and the morphology of the chromatin, as revealed by DAPI staining, was abnormal. Although a few of these worms produced sperm, they did not develop a normal oocyte (Fig. 1). We conclude that puf-8 and tcer-1 function redundantly to ensure normal levels of germ cell proliferation.


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 functions redundantly with tcer-1. Dissected C. elegans adult gonads of the indicated genotype stained with DAPI. Left, distal; right, proximal. The three images are at identical magnification. The double-mutant gonad is significantly smaller than either single mutant and it contains fewer germ cells and no gametes. Scale bars: 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: puf-8 functions redundantly with tcer-1. Dissected C. elegans adult gonads of the indicated genotype stained with DAPI. Left, distal; right, proximal. The three images are at identical magnification. The double-mutant gonad is significantly smaller than either single mutant and it contains fewer germ cells and no gametes. Scale bars: 25 μm.
Mentions: To identify gene(s) that potentially compensate for the lack of PUF-8 at 20°C, a synthetic genetic screen was carried out on the puf-8(+/-) background (Vaid et al., 2013). To map one of the synthetic mutants identified in that screen, we performed an RNAi screen of genes present in a limited genetic interval on chromosome II on the puf-8(zh17) genetic background, which is a strong loss-of-function allele. This RNAi screen identified tcer-1 as functionally redundant with puf-8. Consistent with the earlier results, 100% of the puf-8(zh17) worms were fertile at 20°C (n=150). Similarly, all of the tcer-1(RNAi) worms were fertile and did not show any observable defects (n=500). By contrast, the puf-8(zh17) tcer-1(RNAi) double-mutant worms were 100% sterile at 20°C (n=500). Subsequently, we confirmed the RNAi results by generating a puf-8(-) tcer-1(-) genetic double-mutant strain using tcer-1(tm1452), which has a 392 bp deletion that includes most of the second exon of tcer-1. Similar to the tcer-1(RNAi) worms, tcer-1(tm1452) worms were normal (n=500). In the double-mutant worms, the total number of germ cells was severely reduced and the morphology of the chromatin, as revealed by DAPI staining, was abnormal. Although a few of these worms produced sperm, they did not develop a normal oocyte (Fig. 1). We conclude that puf-8 and tcer-1 function redundantly to ensure normal levels of germ cell proliferation.

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