spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00486

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marin, V. A.
Right arrow Articles by Evans, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marin, V. A.
Right arrow Articles by Evans, T. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

Veronica A. Marin1 and Thomas C. Evans1,2,*

1 Program in Molecular Biology
2 Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262, USA



View larger version (52K):

[in a new window]
  		Fig. 1. The Spatial Control Region of the glp-1 3' UTR is sufficient for translational control in the embryo. (A) A diagram of a single hermaphrodite gonad arm. Mitotic germ cells proliferate near the distal end, and as they move from this region they enter meiosis, differentiate into oocytes, and are then fertilized. GLP-1 protein (in red) is expressed in mitotic germ cells and in anterior cells of the early embryo, but glp-1 mRNA is found throughout (blue lines). (B) Schematic of the glp-1 3' UTR and a chimeric unc-54 3' UTR used in lacZ reporter mRNAs. lacZ coding sequences include a nuclear localization signal. (C) Whole mount of a hermaphrodite injected with lacunc mRNA (no glp-1 sequences) and stained with X-gal. ß-gal activity (dark nuclear stain) was detected in the distal arm (arrows) and in oocyte nuclei (arrowheads), and weakly in embryos (asterisks). The site of injection is indicated by the large arrow. Injected lacZ mRNAs are typically excluded from the distal tip region for unknown reasons (data not shown). (D) A whole mount of a hermaphrodite injected with lacunc(34WT) mRNA. ß-gal activity was not detected in the distal arm (arrows) or in oocytes (arrowheads), but was strongly detected in embryos older than the 4-cell stage (asterisks). (E) An 8-cell embryo from an animal injected with lacunc(34WT) mRNA stained with X-gal and DAPI. Dark ß-gal staining was seen in the four anterior (AB) nuclei; one AB nucleus is not in focus. Staining was not detected or was very weak in four posterior cells (small arrowheads). (F) Whole mount hermaphrodite injected with lacunc(34LS1) mRNA which contains a mutation in the GRE of the SCR (see Fig. 2). Staining was detected in the distal arm (large arrow), near the gonad bend (small arrow), in oocytes (arrowheads), and in some older embryos (not shown).

 


View larger version (40K):

[in a new window]
  		Fig. 5. GLD-1 depletion disrupts regulation of endogenous GLP-1 expression. Animals were injected with gld-1 dsRNA and then stained for GLP-1 protein by immunofluorescence after incubation for 15 hours at 25°C. Gonads are outlined in A and B. (A) A control gonad dissected from a non-injected animal. GLP-1 staining was restricted to the mitotic proliferation zone of the germline (white bracket). (B) A gonad dissected from a gld-1(RNAi) animal. GLP-1 staining extends beyond the mitotic region (bracket) of the distal arm. This gonad also contained oocytes (arrowheads) with diakinesis nuclei, as judged by DAPI staining (not shown). (C) Quantitation of the expansion of GLP-1 staining from the distal tip, following gld-1(RNAi). The number of nuclei were counted from the distal tip to where GLP-1 staining was lost from peripheral membranes in individual gonads. Numbers represent mean±s.d. from three experiments (n=8-17 gonads for each experiment). (D) A 4-cell embryo from a non-injected control animal. (E) A 4-cell embryo from a gld-1(RNAi) animal. The two posterior blastomeres are indicated (arrowheads) in D and E.

 


View larger version (66K):

[in a new window]
  		Fig. 2. Mutational analysis of the glp-1 SCR. (A) Base substitution mutations were made within the full-length glp-1 3' UTR in lacZ reporter mRNAs. The 34 nt sub-region of the SCR is shown, and mutations are underlined. The location of the GRE and GDE elements suggested by the data in (B-F) and in Table 1 are shown at the top; arrowheads indicate that these elements may overlap and/or contain additional untested nucleotides. (B) A 4-cell embryo from an animal injected with lacZ mRNA carrying the wild-type glp-1 3' UTR [lacglp(WT)] and stained for ß-gal. Dark staining can be seen in the anterior cells ABa and ABp, but not in posterior cells (arrowheads). (C) A 4-cell embryo from an animal injected with lacglp(LS1) mRNA. Dark staining can be seen in all four cells. (D) A whole mount of a hermaphrodite injected with lacglp(LS2) mRNA; no staining was detected in the distal arm (bracket), oocytes (arrowheads), or embryos (asterisks). (E-G) In situ hybridization to detect injected lacZ reporter mRNA in embryos with a lacZ probe. (E) A 2-cell embryo carrying lacglp(WT) mRNA. (F) A 4-cell embryo carrying lacglp(LS2) mRNA. (G) A 4-cell embryo from a non-injected animal.

 


View larger version (79K):

[in a new window]
  		Fig. 3. GLD-1 is pulled out of crude extracts by tagged SCR RNA. (A) Proteins were pulled out of crude extracts using digoxigenin (dig)-labeled RNAs that included the entire 61 nt glp-1 SCR (total RNA size, including vector sequence, was 104 nt). Precipitated proteins were subjected to UV cross-linking to a 32P-labeled RNA containing 34 nt of the wild-type glp-1 SCR (the GRE/GDE region in Fig. 2; total RNA probe was 68 nt). 58 and 30 kDa (p58 and p30) proteins labeled by the probe are indicated. Dig-labeled RNAs were either wild type, had the M7 mutation in the GRE and GDE (see Fig. 2), or a 13 nt mutation (M9) at the 3' end of the SCR, downstream of the 34 nt GRE/GDE region (not shown). (B) Proteins pulled out by wild-type dig-SCR RNA were UV cross-linked to 32P-labeled probes that had 34 nt of wild-type or mutant SCR sequences (refer to Fig. 2). The ratios of p58 to p30 varied from prep to prep, possibly due to proteolysis (data not shown). (C) Competition of wild-type 32P-labeled probe by unlabeled wild-type or mutant RNAs, as assayed by UV cross-linking. Unlabeled RNAs were added at 10- and 500-fold molar excess of labeled probe. Lanes 1 and 8 have no competitor RNA added. (D) A western blot of proteins pulled out by dig-tagged wild-type (WT), M7 or M9 SCR RNAs (71 nt) probed with GLD-1 antibodies. Protein samples used for this blot were from the same samples as used for UV cross-linking in A. The arrow marks the position of the single band detected in total worm homogenate (data not shown).

 


View larger version (18K):

[in a new window]
  		Fig. 4. Recombinant GLD-1 binds directly and specifically to the GRE of glp-1 RNA. (A) GST pull down of radiolabeled glp-1 34 nt RNAs by GST-GLD-1. Shown are means of two binding reactions±the variance from a single experiment; similar results were seen in three separate experiments. (B) Competitive displacement of GST GLD-1 from 32P-labeled SCR (34 nt) with unlabeled wild-type or mutant RNAs. A filter-binding assay was used to detect GLD-1 binding to wild-type 32P-labeled 34 nt RNA that contains both the GRE and GDE. Increasing concentrations of unlabeled 34 nt RNAs were added to the binding reactions. 100% binding is the amount bound in absence of any competitor. Shown is the mean±s.d. of three to four separate binding reactions.

 


View larger version (9K):

[in a new window]
  		Fig. 6. A model for GLD-1 and glp-1 mRNA regulation in the early embryo. In posterior cells, GLD-1 binds to the GRE, probably with other factors (not shown), and represses glp-1 mRNA translation. In anterior cells, a derepressor (D) binds to the GDE to inhibit GLD-1 binding or activity, leading to glp-1 translation. Localization of glp-1 translation is further ensured by enrichment of GLD-1 in posterior cells.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003