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First published online 14 November 2006
doi: 10.1242/jcs.03287

Journal of Cell Science 119, 4964-4973 (2006)
Published by The Company of Biologists 2006
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The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules

Philip Becht*, Julian König* and Michael Feldbrügge{ddagger}

Max Planck Institute for Terrestrial Microbiology, Department for Organismic Interactions, Karl-von-Frisch-Str., 35043 Marburg, Germany


Figure 1
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  		Fig. 1. Loss of Rrm4 causes polarity defects in filaments. (A) Colonies of AB33 and derivatives 24 hours after filament induction on charcoal-containing minimal medium plates (respective rrm4 alleles labelled at the top). (B) Filament formation was elicited in strains AB33, AB33rrm4{Delta} and AB33rrm4G for 4 hours in nitrate-containing minimal medium resulting in the nar1-promoter-mediated expression of an active bW2/bE1 heterodimer. Bar, 10 µm. (C) Graph indicating the percentage of filaments with empty sections 6 hours after filament induction. More than 40 filaments were analysed each (relevant rrm4 alleles and number of transformant below). (D) Graph (labelling as in C) indicating the percentage of cells that grew bipolarly 4 hours after filament induction. (E) Graph (labelling as in C) showing the length of the living tip cells of AB33 and derivatives measured 6 hours after filament induction (error bars indicate s.d., more than ten filaments were analysed for each strain).

 

Figure 2
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  		Fig. 2. Rrm4GT binds RNA in vivo probably via N-terminal RRMs. (A) ELAV-like protein Rrm4 contains a unique domain architecture. Domain organisation of RRM-containing proteins is depicted schematically. RRM and PABC domains are indicated as rectangles and ovals, respectively. Accession numbers: ELAV, D. melanogaster; P16914; Pab1p, S. cerevisiae; P04147; Rrm4, U. maydis; UM10836 (http://mips.gsf.de/genre/proj/ustilago/). (B) Sequence comparisons of RRMs and PABC domains are given. The conserved RNP1 and RNP2 regions of RRM proteins Rrm4 from U. maydis as well as ELAV and Sex-lethal (Sxl) from D. melanogaster are compared (accession numbers UM10836, http://mips.gsf.de/genre/proj/ustilago/, P16914 and Q24668, respectively). Numbers given in small and large fonts indicate amino acid positions and distance between RNP1 and RNP2, respectively. To generate loss-of-function mutants, the boxed sequence in the RNP1 regions of RRM1 and RRM3 were mutated to alanine. Below, the conserved core sequence of the Rrm4 PABC domain is compared with those of poly(A)-binding proteins from human and S. cerevisiae (accession numbers UM10836, AAH23520, and AAA34838, respectively). Amino acids above the Rrm4 sequence indicate point mutations in critical amino acids shown to be structural and functionally important in the human PABC domain (Kozlov et al., 2004Go). Dark and light shading indicate identical and similar amino acids, respectively. (C) Modified CLIP analysis of AB33rrm4GT filaments 6 hours after induction. Covalently bound RNA is detected as radioactively labelled protein-RNA complexes larger in size than Rrm4GT (117 kD after TEV cleavage; size markers are shown on the left; asterisk, unspecific band). Increasing amounts of RNase T1 treatment (lanes 3-4; ++ indicates 100-fold increase) before labelling reduces the size of the bound RNA resulting in a sharper band. RNase A treatment after labelling (lanes 5-8) removes the radioactive signal indicating that the crosslinked material is RNA. Equal loading was verified by western blotting using anti-GFP antibodies on the same membrane (bottom). (D) Western blot experiments using anti-GFP antibodies showing equal protein amounts of Rrm4G and Rrm4GT detected at the onset (0) or after 6 hours of filamentation. Equal loading was verified using anti-{alpha}-tubulin antibodies (size markers are shown in kDa on the left). (E) CLIP experiments as in C using AB33rrm4GT (0 and 6 hours after filament induction) and variants expressing mutant alleles rrm4GTmP (mP), rrm4GTmR1 (mR1) and rrm4GTmR3 (mR3) (6 hours after filament induction). Relative RNA binding (%) was quantified after phosphoimaging.

 

Figure 3
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  		Fig. 3. Rrm4G is assembled into cytoplasmic particles that shuttle along cytoskeletal tracks. (A) AB33rrm4G filaments are shown 4 hours after induction. Epifluorescence images are taken from time-lapse Movie 1 in supplementary material. Rectangles indicate regions that are magnified below. Arrows indicate particles that move along cytoskeletal tracks at the distal pole (left panels) and at the tip (right panels; time in seconds). Bars, 5 µm. (B) Whisker diagram indicating density of Rrm4G-containing particles (particles/µm) in strain AB33rrm4G measured at the onset or after 6 hours of filamentation. The top and bottom of each rectangle represent the 75th and 25th percentiles, respectively. The median is the horizontal line within the box, and the top and bottom borders of the whiskers show the 95th and 5th percentiles, respectively. More than 200 particles were analysed for each strain. The number of the transformant is given below. (C) Whisker diagram (labelling as in B) showing the relative fluorescence intensity of Rrm4G-containing particles in strain AB33rrm4G measured at the onset or after 6 hours of filamentation. More than 80 particles each were analysed (number of transformant is indicated below).

 

Figure 4
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  		Fig. 4. The N-terminal RRM1 and the C-terminal PABC domain are important for function. (A) Graph indicating the percentage of filaments with empty sections 4 hours after filament induction (labelling as in Fig. 1C). The mutated alleles of rrm4 in AB33 derivatives (Fig. 2B) and the number of transformant are given below. More than 40 filaments were analysed for each group. (B) Graph showing the percentage of filaments that grew bipolarly 4 hours after induction (labelling as in A). For comparison the data for rrm4 and rrm4{Delta} (Fig. 1C,D) are given as dashed lines. (C) Filaments of AB33 derivatives 4 hours after induction. Bar, 5 µm. Frames are taken from supplementary material Movies 2 and 3. To document motility of particles, overlays of two frames that are 1 second apart are shown. The first and second frames are coloured in green and red, respectively, indicating non-motile particles in yellow (top, mutations in PABC) and moving particles in green and red (right and bottom, mutations in RRM). (D) Whisker diagram (labelling as in Fig. 3B) indicating the relative fluorescence intensity of Rrm4G-containing particles in strains AB33rrm4GmR1 and AB33rrm4GmR3 measured at the onset or after 6 hours of filamentation. For comparison, medians from the data obtained at 0 and 6 hours of filamentation (lower and upper dashed lines, respectively) from AB33rrm4G#1 (data from Fig. 3C) are shown. More than 80 particles were analysed for each group (number of transformant is indicated).

 

Figure 5
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  		Fig. 5. Rrm4-containing particles shuttle in vivo along microtubules. (A) Filaments of AB33rrm4G were treated with benomyl 4 hours after filament induction and time-lapse movies were taken at the onset (0 seconds, top) and after 23 minutes (bottom) of treatment. Overlays of two frames 1 second apart (coloured in green and red) from supplementary material Movie 4 are shown (moving particles in green and red, top; non-motile particles in yellow, bottom). Bar, 10 µm. (B) In vivo co-localisation of Rrm4R-containing particles (red, arrows) with dynamic Tub1G-decorated microtubules (green) in AB33rrm4R/tub1G filaments 4 hours after induction. Bar, 5 µm. Frames are from supplementary material Movie 5. Rectangles indicate regions that are magnified below. Bar, 1 µm. Time is indicated in seconds.

 

Figure 6
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  		Fig. 6. The loss of Kin1 causes defects in polarity and shuttling of Rrm4G-containing particles. (A) Graph indicating the percentage of filaments that grow bipolarly 4, 6 and 10 hours after induction. More than 30 filaments were analysed for each strain (number of transformant is given below). (B) AB33rrm4G/kin1{Delta} filaments 6 hours after induction. Epifluorescence image is taken from time-lapse Movie 6 in supplementary material. In bipolar filaments, Rrm4G-containing particles accumulate at the poles. Bar, 5 µm.

 

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