QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 June 2004
doi: 10.1242/dev.01196

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mootz, D. Articles by Hunter, C. P. Search for Related Content PubMed PubMed Citation Articles by Mootz, D. Articles by Hunter, C. P. Social Bookmarking                         What's this?

The STAR/Maxi-KH domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1

Darcy Mootz, Diana M. Ho^* and Craig P. Hunter

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

View larger version (45K): [in a new window]   Fig. 1. Multiple regions of the pal-1 3' UTR repress translation in the distal gonad arm. (A) Schematic of an adult gonad arm illustrating the progressive maturation of germ cells. pal-1 mRNA (yellow) is present throughout the germline, yet PAL-1 protein (red) is detectable only in immature oocytes (47% of gonad arms (n=49) exhibit no PAL-1 expression). (B-D) Gonad arms injected with lacZ, lacZ::3' UTR and lacZ::GRE RNA, respectively, and stained with X-gal. Insets show dissected distal gonad arms that were injected with the same RNAs (except in D, where lacZ::2X GRE RNA was used) and hybridized with lacZ antisense probe. (E) Schematics of lacZ reporter constructs with blue bars denoting the percentage of injected gonad arms with distal germline expression. Regions A, B, and the GRE mediate repression. (F) Sequence of the GRE.

View larger version (45K): [in a new window]   Fig. 2. gld-1 mediates the translational repression activity of the GRE. The distal gonad arms are outlined and the autofluorescent intestine is visible in A and C. (A) GH::pie-1 3' UTR is expressed in nuclei throughout the proximal and distal germline. (B) GH::2X GRE is expressed in proximal germline nuclei, but not distal germline nuclei. (C) GH::2X GRE is ectopically expressed in distal germline nuclei following gld-1 RNAi. The characteristic gld-1(–) proximal germline tumor is evident. (D) Multi-second exposure of a dissected GH::2X GRE gonad arm. Weak expression is observed in the distal germline cytoplasm. (E) RNase protection assays with pal-1 and gfp probes and 60 µg of total RNA from wild-type or gld-1 (RNAi) GH::2X GRE L4 larvae (integrated transgene). gld-1 does not destabilize pal-1 or GH::2X GRE mRNA.

View larger version (56K): [in a new window]   Fig. 3. gld-1 represses the distal germline expression of both PAL-1 and MEX-3. The distal gonad arms are outlined. (A,B,D,E) Anti-PAL-1 (A,B) and anti-MEX-3 (D,E) staining in wild-type (A,D) and gld-1(RNAi) (B,E) gonad arms. (A,D) No expression is detected in the distal germline. PAL-1 is detectable in immature oocytes in 53% of gonad arms (n=49) and the staining shown here is an extreme example illustrating the maximal PAL-1 expression observed. (B,E) Ectopic expression is observed in the distal germline. (C) Anti-PAL-1 staining in a gld-1 (RNAi); mex-3 (RNAi) gonad arm. Compared to gld-1 RNAi worms, significantly more ectopic PAL-1 is observed in the distal germline (see text for quantitation).

View larger version (36K): [in a new window]   Fig. 4. GLD-1 selectively binds pal-1 3' UTR elements that mediate germline repression in vivo. Biotinylated 3' UTR RNA fragments were incubated with extracts prepared from adults expressing a rescuing GLD-1::GFP::FLAG fusion protein (GGF). Biotinylated RNA was isolated using streptavidin magnetic beads and bound proteins were subjected to SDS-PAGE and western analysis with anti-FLAG antibody. S, sense RNA; AS, antisense RNA. This assay shows the expected specificity, as GGF is captured by tra-2 3' UTR RNA that bears GLD-1 binding sites, but not by a tra-2 antisense RNA. 2X GRE, A, and B RNAs capture GGF, while antisense 2X GRE and C RNAs do not, showing that binding activity in extracts correlates with in vivo repression activity.

View larger version (38K): [in a new window]   Fig. 5. pal-1 mRNA and GLD-1 protein co-fractionate with ribosomes. (A) RNase protection assays with pal-1 probe and 60 µg of yeast RNA or RNA from wild-type and glp-4 (bn2ts) L4 larvae and adults. Densitometry of two replicates indicates that approximately 75% of pal-1 mRNA from wild-type L4s and 95% of pal-1 mRNA from adults is associated with the germline. (B) A representative histogram illustrating the total RNA in 25 0.5 ml fractions taken manually after worm extracts were fractionated by sucrose density gradient sedimentation. Inset shows the absorbance profile when 56 0.2 ml fractions were taken from a gradient run under the same conditions. The fractions containing ribosomes are indicated in red and bars below the graph indicate the fractions pooled for RNase protection assays. Both pal-1 and mex-3 mRNA co-fractionate with ribosomes. Y, control containing only yeast RNA. (C) Metrizamide equilibrium sedimentation of L4 larvae. Curve denotes the refractive index of the gradient fractions, while the bars denote the percentage of total RNA recovered in the pooled fractions. RNase protection assays on pooled fractions indicate that pal-1 mRNA is found at the predicted refractive index for C. elegans ribosomes under these conditions (1.412, indicated by dotted line and red bar). The mex-3 probe shown in the input lane was not included in the RNase protection assays. (D) Distribution of GLD-1 protein from adult hermaphrodites following metrizamide sedimentation. The index of refraction is indicated above each lane. Western analysis with anti-GLD-1 antibody indicates that GLD-1 is enriched in the predicted ribosome fraction (red).

View larger version (52K): [in a new window]   Fig. 6. gld-1 represses the distal germline expression of SPN-4 and MEX5/6. The distal gonad arms are outlined. (A,B) Anti-SPN-4 and (C,D) anti-MEX5/6 staining in wild-type (A,C) and gld-1(q485) (B,D) gonad arms. Expression is not detected in the distal germline of wild type (A,C) but ectopic expression is observed in gld-1(q485) (B,D).

View larger version (28K): [in a new window]   Fig. 7. Proposed model for PAL-1 repression in the germline. (A) GLD-1 represses the translation of both pal-1 and mex-3 in the distal gonad arm, while MEX-3 represses pal-1 translation in the proximal gonad arm. GLD-1 and MEX-3, shown in blue and green respectively, have graded expression patterns in the gonad, with lowest levels found in the bend. A leaky switch from GLD-1- to MEX-3-mediated repression may account for the restricted PAL-1 expression (red) in the bend of the gonad arm. (B) GLD-1 binds the GRE and may repress pal-1 translation after ribosome loading.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter