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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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The levels of several germline mRNAs are dependent on PUF-8 and TCER-1. (A) Mutant to wild type ratios of the indicated mRNA levels quantitated by RT-PCR (see Materials and methods). (B) Comparison of the levels of the spliced (mRNA) and unspliced (pre-mRNA) versions of the indicated mRNAs in the puf-8(-) tcer-1(-) double mutant. ***P=0.0003; **P=0.0038; *P=0.0344; Student’s t-test, mRNA versus pre-mRNA levels. (C) Comparison of mRNA/pre-mRNA ratio between the wild type and the puf-8(-) tcer-1(-) double mutant. This ratio is independent of overall RNA levels, and thus eliminates potential inaccuracies arising from variations in the overall RNA levels between the wild type and double mutant. ***P=0.0001; **P=0.003; Student’s t-test. Error bars indicate s.d.
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Figure 3: The levels of several germline mRNAs are dependent on PUF-8 and TCER-1. (A) Mutant to wild type ratios of the indicated mRNA levels quantitated by RT-PCR (see Materials and methods). (B) Comparison of the levels of the spliced (mRNA) and unspliced (pre-mRNA) versions of the indicated mRNAs in the puf-8(-) tcer-1(-) double mutant. ***P=0.0003; **P=0.0038; *P=0.0344; Student’s t-test, mRNA versus pre-mRNA levels. (C) Comparison of mRNA/pre-mRNA ratio between the wild type and the puf-8(-) tcer-1(-) double mutant. This ratio is independent of overall RNA levels, and thus eliminates potential inaccuracies arising from variations in the overall RNA levels between the wild type and double mutant. ***P=0.0001; **P=0.003; Student’s t-test. Error bars indicate s.d.

Mentions: As shown in Fig. 3, in the puf-8(-) tcer-1(-) double mutant, the levels of six of the 16 mRNAs that we measured, namely egg-5, pal-1, cyb-2.1, pos-1, spn-4 and oma-1 mRNAs, were only ∼10-30% of the wild type. By contrast, the pre-mRNA levels of all but one of them - the unspliced version of oma-1 mRNA could not be amplified - showed a ∼2- to 3-fold increase in the double mutant compared with the wild type. The levels of the unspliced and spliced forms of these mRNAs did not vary significantly between the wild type and single mutants. These results show that PUF-8 and TCER-1 function redundantly to maintain normal levels of the spliced form of these mRNAs.


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)

The levels of several germline mRNAs are dependent on PUF-8 and TCER-1. (A) Mutant to wild type ratios of the indicated mRNA levels quantitated by RT-PCR (see Materials and methods). (B) Comparison of the levels of the spliced (mRNA) and unspliced (pre-mRNA) versions of the indicated mRNAs in the puf-8(-) tcer-1(-) double mutant. ***P=0.0003; **P=0.0038; *P=0.0344; Student’s t-test, mRNA versus pre-mRNA levels. (C) Comparison of mRNA/pre-mRNA ratio between the wild type and the puf-8(-) tcer-1(-) double mutant. This ratio is independent of overall RNA levels, and thus eliminates potential inaccuracies arising from variations in the overall RNA levels between the wild type and double mutant. ***P=0.0001; **P=0.003; Student’s t-test. Error bars indicate s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The levels of several germline mRNAs are dependent on PUF-8 and TCER-1. (A) Mutant to wild type ratios of the indicated mRNA levels quantitated by RT-PCR (see Materials and methods). (B) Comparison of the levels of the spliced (mRNA) and unspliced (pre-mRNA) versions of the indicated mRNAs in the puf-8(-) tcer-1(-) double mutant. ***P=0.0003; **P=0.0038; *P=0.0344; Student’s t-test, mRNA versus pre-mRNA levels. (C) Comparison of mRNA/pre-mRNA ratio between the wild type and the puf-8(-) tcer-1(-) double mutant. This ratio is independent of overall RNA levels, and thus eliminates potential inaccuracies arising from variations in the overall RNA levels between the wild type and double mutant. ***P=0.0001; **P=0.003; Student’s t-test. Error bars indicate s.d.
Mentions: As shown in Fig. 3, in the puf-8(-) tcer-1(-) double mutant, the levels of six of the 16 mRNAs that we measured, namely egg-5, pal-1, cyb-2.1, pos-1, spn-4 and oma-1 mRNAs, were only ∼10-30% of the wild type. By contrast, the pre-mRNA levels of all but one of them - the unspliced version of oma-1 mRNA could not be amplified - showed a ∼2- to 3-fold increase in the double mutant compared with the wild type. The levels of the unspliced and spliced forms of these mRNAs did not vary significantly between the wild type and single mutants. These results show that PUF-8 and TCER-1 function redundantly to maintain normal levels of the spliced form of these mRNAs.

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