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Translational control of the oogenic program by components of OMA ribonucleoprotein particles in Caenorhabditis elegans.

Spike CA, Coetzee D, Nishi Y, Guven-Ozkan T, Oldenbroek M, Yamamoto I, Lin R, Greenstein D - Genetics (2014)

Bottom Line: Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3'-untranslated regions (3'-UTRs).Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2.We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Minneapolis, Minnesota 55455.

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LIN-41 represses the translation of OMA targets in oocytes. (A–H) lin-41(RNAi) (B, D, F, and H) strongly enhances GFP::H2B expression from reporter transgenes containing the zif-1 (A and B), cdc-25.3 (C and D), or rnp-1 (E and F) 3′-UTRs in oocytes. There was no apparent increase in GFP::H2B expression from the reporter transgene containing the control fbf-2 3′-UTR (G and H). (I and J) GFP::H2B expression from the zif-1 reporter transgene can be seen in the oocytes of fertile lin-41(ma104) animals (67%; n = 54), but not wild-type controls (0%; n > 36). (K–N) GFP::H2B expression from the cdc-25.3 (K and L) and zif-1 (M and N) reporter transgenes is increased in the oocytes of lin-41(tn1487ts) mutants raised at 25° relative to controls. (O and P) Expression from the zif-1 reporter transgene (green) begins as lin-41(n2914) germ cells start to develop into oocytes (O); the most proximal oocyte is GFP positive (arrowhead). The germ line of this lin-41(n2914) animal was imaged just prior to vulval eversion (P), which would normally occur around the time of the L4-to-adult molt (Sharma-Kishore et al. 1999). Identical exposure times were used to collect the paired images with the exception that the control in A was 40% overexposed compared to B. Bar, 20 μm.
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fig8: LIN-41 represses the translation of OMA targets in oocytes. (A–H) lin-41(RNAi) (B, D, F, and H) strongly enhances GFP::H2B expression from reporter transgenes containing the zif-1 (A and B), cdc-25.3 (C and D), or rnp-1 (E and F) 3′-UTRs in oocytes. There was no apparent increase in GFP::H2B expression from the reporter transgene containing the control fbf-2 3′-UTR (G and H). (I and J) GFP::H2B expression from the zif-1 reporter transgene can be seen in the oocytes of fertile lin-41(ma104) animals (67%; n = 54), but not wild-type controls (0%; n > 36). (K–N) GFP::H2B expression from the cdc-25.3 (K and L) and zif-1 (M and N) reporter transgenes is increased in the oocytes of lin-41(tn1487ts) mutants raised at 25° relative to controls. (O and P) Expression from the zif-1 reporter transgene (green) begins as lin-41(n2914) germ cells start to develop into oocytes (O); the most proximal oocyte is GFP positive (arrowhead). The germ line of this lin-41(n2914) animal was imaged just prior to vulval eversion (P), which would normally occur around the time of the L4-to-adult molt (Sharma-Kishore et al. 1999). Identical exposure times were used to collect the paired images with the exception that the control in A was 40% overexposed compared to B. Bar, 20 μm.

Mentions: Mass spectrometry is extremely sensitive, and >250 different proteins were identified by at least two peptides in both OMA-1 RNP purifications (Figure S8, File S2). Many of these proteins, like CGH-1 and CAR-1, have RNA-related functions and could be important components of OMA-1 RNPs in vivo. Other copurifying proteins likely represent abundant contaminants (e.g., UNC-54/myosin and VIT-1/vitellogenin; see Materials and Methods). We focused on the subset of proteins that copurify with OMA-1 from fog-1(ts) females after RNase treatment based on the expectation that close associations with OMA-1 (be they direct or indirect) might be at least partially resistant to RNase treatment. Many proteins, including some with RNA-related functions (e.g., CGH-1, EDC-3, and CEY-4), were depleted from OMA-1 RNPs by RNase treatment, leaving a much smaller pool of candidates (133 different proteins; Figure S8). Importantly, the eIF4E-binding protein IFET-1 continued to copurify with OMA-1 in the presence of RNase A (File S2). Prior work established that IFET-1 interacts with OMA-1in vitro and represses the translation of OMA target 3′-UTR reporters in vivo (Li et al. 2009; Guven-Ozkan et al. 2010; Oldenbroek et al. 2013). Next, proteins identified in negative controls or as abundant contaminants were excluded from consideration, leaving a smaller list of OMA-1-associated proteins (51 different proteins; Figure S8). This step eliminated CAR-1, but again retained IFET-1 (File S2). It is difficult to eliminate all contaminants identified by mass spectrometry using a limited number of negative controls (Mellacheruvu et al. 2013), so we examined the biological functions of the remaining proteins in more detail (Table 1, File S2).


Translational control of the oogenic program by components of OMA ribonucleoprotein particles in Caenorhabditis elegans.

Spike CA, Coetzee D, Nishi Y, Guven-Ozkan T, Oldenbroek M, Yamamoto I, Lin R, Greenstein D - Genetics (2014)

LIN-41 represses the translation of OMA targets in oocytes. (A–H) lin-41(RNAi) (B, D, F, and H) strongly enhances GFP::H2B expression from reporter transgenes containing the zif-1 (A and B), cdc-25.3 (C and D), or rnp-1 (E and F) 3′-UTRs in oocytes. There was no apparent increase in GFP::H2B expression from the reporter transgene containing the control fbf-2 3′-UTR (G and H). (I and J) GFP::H2B expression from the zif-1 reporter transgene can be seen in the oocytes of fertile lin-41(ma104) animals (67%; n = 54), but not wild-type controls (0%; n > 36). (K–N) GFP::H2B expression from the cdc-25.3 (K and L) and zif-1 (M and N) reporter transgenes is increased in the oocytes of lin-41(tn1487ts) mutants raised at 25° relative to controls. (O and P) Expression from the zif-1 reporter transgene (green) begins as lin-41(n2914) germ cells start to develop into oocytes (O); the most proximal oocyte is GFP positive (arrowhead). The germ line of this lin-41(n2914) animal was imaged just prior to vulval eversion (P), which would normally occur around the time of the L4-to-adult molt (Sharma-Kishore et al. 1999). Identical exposure times were used to collect the paired images with the exception that the control in A was 40% overexposed compared to B. Bar, 20 μm.
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Related In: Results  -  Collection

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fig8: LIN-41 represses the translation of OMA targets in oocytes. (A–H) lin-41(RNAi) (B, D, F, and H) strongly enhances GFP::H2B expression from reporter transgenes containing the zif-1 (A and B), cdc-25.3 (C and D), or rnp-1 (E and F) 3′-UTRs in oocytes. There was no apparent increase in GFP::H2B expression from the reporter transgene containing the control fbf-2 3′-UTR (G and H). (I and J) GFP::H2B expression from the zif-1 reporter transgene can be seen in the oocytes of fertile lin-41(ma104) animals (67%; n = 54), but not wild-type controls (0%; n > 36). (K–N) GFP::H2B expression from the cdc-25.3 (K and L) and zif-1 (M and N) reporter transgenes is increased in the oocytes of lin-41(tn1487ts) mutants raised at 25° relative to controls. (O and P) Expression from the zif-1 reporter transgene (green) begins as lin-41(n2914) germ cells start to develop into oocytes (O); the most proximal oocyte is GFP positive (arrowhead). The germ line of this lin-41(n2914) animal was imaged just prior to vulval eversion (P), which would normally occur around the time of the L4-to-adult molt (Sharma-Kishore et al. 1999). Identical exposure times were used to collect the paired images with the exception that the control in A was 40% overexposed compared to B. Bar, 20 μm.
Mentions: Mass spectrometry is extremely sensitive, and >250 different proteins were identified by at least two peptides in both OMA-1 RNP purifications (Figure S8, File S2). Many of these proteins, like CGH-1 and CAR-1, have RNA-related functions and could be important components of OMA-1 RNPs in vivo. Other copurifying proteins likely represent abundant contaminants (e.g., UNC-54/myosin and VIT-1/vitellogenin; see Materials and Methods). We focused on the subset of proteins that copurify with OMA-1 from fog-1(ts) females after RNase treatment based on the expectation that close associations with OMA-1 (be they direct or indirect) might be at least partially resistant to RNase treatment. Many proteins, including some with RNA-related functions (e.g., CGH-1, EDC-3, and CEY-4), were depleted from OMA-1 RNPs by RNase treatment, leaving a much smaller pool of candidates (133 different proteins; Figure S8). Importantly, the eIF4E-binding protein IFET-1 continued to copurify with OMA-1 in the presence of RNase A (File S2). Prior work established that IFET-1 interacts with OMA-1in vitro and represses the translation of OMA target 3′-UTR reporters in vivo (Li et al. 2009; Guven-Ozkan et al. 2010; Oldenbroek et al. 2013). Next, proteins identified in negative controls or as abundant contaminants were excluded from consideration, leaving a smaller list of OMA-1-associated proteins (51 different proteins; Figure S8). This step eliminated CAR-1, but again retained IFET-1 (File S2). It is difficult to eliminate all contaminants identified by mass spectrometry using a limited number of negative controls (Mellacheruvu et al. 2013), so we examined the biological functions of the remaining proteins in more detail (Table 1, File S2).

Bottom Line: Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3'-untranslated regions (3'-UTRs).Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2.We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Minneapolis, Minnesota 55455.

Show MeSH
Related in: MedlinePlus