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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

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 Moy, T. I.
Right arrow Articles by Silver, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moy, T. I.
Right arrow Articles by Silver, P. A.
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?

Requirements for the nuclear export of the small ribosomal subunit

Terence I. Moy1 and Pamela A. Silver2,*

1 Department of Molecular Biology, Massachusetts General Hospital, 50 Blossum Street, Boston, MA 02114, USA
2 The Dana-Farber Cancer Institute, 1 Jimmy Fund Way, Boston, MA 02115, USA



View larger version (34K):

[in a new window]
  		Fig. 1. The localization pattern of 5' ITS1 from the small ribosomal subunit export assay. After the small ribosomal subunit is exported from the nucleus, the 5' ITS1 RNA is cleaved away from the 18S rRNA in the cytoplasm and then degraded by the exonuclease Xrn1. Cells lacking XRN1 accumulate 5' ITS1 in the cytoplasm as long as ribosome assembly and export occurs (a). A xrn1{Delta} mutant defective in small ribosomal subunit assembly accumulates 5' ITS1 to the nucleolus, the site of rRNA transcription and processing (c). 5' ITS1 also localizes to the nucleolus of wild-type XRN1+ cells (d). A mutant defective in small ribosomal subunit nuclear export accumulates 5' ITS1 throughout the nucleoplasm in both xrn1{Delta} (b) and XRN1+ strain backgrounds (e). Defects in ribosome assembly cannot be detected in XRN1+ strains because 5' ITS1 remains localized to the nucleolus.

 


View larger version (66K):

[in a new window]
  		Fig. 2. CRM1 and YRB2 are required for nuclear export of the small ribosomal subunit. (A) 5' ITS1 accumulates in the nucleoplasm of a crm1 mutant. 5' ITS1 was localized in CRM1 (wild-type, b) and crml(T539C) (e) cells after treatment with leptomycin B for 15 minutes. Chromosomal DNA was labeled with DAPI to identify the location of the nucleus (a,d), and Nomarski optics were used to visualize cell morphology (c,f). Arrows point to DAPI-stained chromatin while arrowheads point to the nucleolus. A magnified view of a cell is shown below the lettered panels. (B) 5' ITS1 accumulates in the nucleoplasm of a yrb2{Delta} mutant. 5' ITS1 was localized in YRB2 (wild-type, b,f) and yrb2{Delta} (d,h) cells grown at 30°C (b,d) or shifted to the restrictive temperature of 15°C (f,h). Chromosomal DNA was labeled with DAPI to identify the location of the nucleus (a,c,e,g). Arrows point to DAPI-stained chromatin, while arrowheads point to the nucleolus. A magnified view of a cell is shown for e, f, g and h. (C) 20S pre-rRNA processing is delayed in yrb2{Delta} cells. YRB2 (lanes 1-6) and yrb2{Delta} (lanes 7-12) cells were shifted to 15°C for 24 hours, pulse-labeled for 5 minutes, and chased for 5, 10, 20, 40 or 60 minutes. RNA was extracted and run on a formaldehyde-agarose gel. Sizes of the rRNAs are indicated.

 


View larger version (20K):

[in a new window]
  		Fig. 3. yrb2{Delta} cells are deficient in the levels of the small ribosomal subunit. (A) YRB2+ and yrb2{Delta} cells were shifted to 15°C for 24 hours, and polysome profiles were resolved in 7-49% sucrose gradients. Abs254nm was monitored continuously and represents the vertical axis of the profiles. The top of the gradient is on the left-hand side of the profile. The positions of free 40S and 60S subunits, 80S ribosomes, and polysomes are indicated. (B) YRB2+ and yrb2{Delta} cells were shifted to 15°C for 24 hours, and ribosomal subunit profiles were resolved in 10-35% sucrose gradients.

 


View larger version (63K):

[in a new window]
  		Fig. 4. Overexpression of YRB2 is toxic. (A) Overexpression of YRB2 causes the mislocalization of 5' ITS1. 5' IST1 was localized in wild-type cells containing pGAL vector (b) or pGAL-YRB2 (d) after 1 hour induction with galactose. Chromosomal DNA was labeled with DAPI (a,c). Arrows point to DAPI-stained chromatin while arrowheads point to the nucleolus. Magnified views of two cells are shown below each panel. (B) High-copy expression of YRB2 inhibits the growth of rat2-1/nup120 cells. rat2-1 (PSY209) or wild-type (PSY580) cells containing 2µ vector (pPS701), full-length YRB2 (pPS2096), N-terminal truncation (pPS2104), deletion of the Nup FG motifs (pPS2105), or the C-terminal/Ran-binding domain truncation (pPS2106) were grown on trp- dropout plates at 25°C.

 


View larger version (74K):

[in a new window]
  		Fig. 5. A shift-back protocol facilitates the detection of ribosome biogenesis defects. (A) 5' ITS1 was localized in xrn1{Delta}, nic96-1 xrn1{Delta}, and kap104-16 xrn1{Delta} after shifting to 37°C for 3 hours (b,f,j, respectively) or shifting back from 37°C to 25°C for 30 minutes (d,h,l). Chromosomal DNA was labeled with DAPI (a,c,e,g,i,k). (B) pre-rRNA processing in wild-type (lanes 1-6) and nic96-1 (lanes 7-12) cells shifted to 37°C or shifted-back to 25°C. Cells were pulse labeled for 3 minutes and chased for 0,3 or 10 minutes. RNA was extracted and run on a formaldehyde-agarose gel. Sizes of the rRNAs are indicated.

 


View larger version (89K):

[in a new window]
  		Fig. 6. Ribosome export mutants identified by screening ts- mutant libraries. (A) 5' ITS1 localization in rat2-1/nup120 (i) grown at 25°C and in wild type (h), rat7-1/nup159 (j), nsp1(L697P) (k), gle2(N273K,D290N) (l), yrb1(F191S) (m), and prp20(S297N) (n) mutants shifted to 37°C for 1 hour. Chromosomal DNA was labeled with DAPI (a-g). Arrows point to DAPI-stained chromatin while arrowheads point to the nucleolus. (B) Pre-rRNA processing in wild-type (lanes 1-3 and 7-9), rat7-1/nup159 (lanes 4-6), nsp1(L697P) (lanes 10-12), gle2(N273K,D290N) (lanes 13-15), and yrb1(F191S) (lanes 16-18) mutants shifted to 37°C for 1 hour. Cells were pulse labeled for 3 minutes (lanes 1-6) or 1 minute (lanes 7-18). Pulse and chase times are indicated above each lane. Sizes of the rRNAs are indicated.

 

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 2002