English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex.

Wolf, E., Kastner, B., & Lührmann, R. (2012). Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex. RNA, 18(7), 1347-1357. doi:10.1261/rna.033910.112.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-A11A-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-823A-9
Genre: Journal Article

Files

show Files
hide Files
:
1479100.pdf (Publisher version), 2MB
Name:
1479100.pdf
Description:
-
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-
:
1479100_SuppFigs.pdf (Supplementary material), 2MB
Name:
1479100_SuppFigs.pdf
Description:
-
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-

Locators

show
hide
Description:
-

Creators

show
hide
 Creators:
Wolf, E.1, Author              
Kastner, B.1, Author              
Lührmann, R.1, Author              
Affiliations:
1Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578576              

Content

show
hide
Free keywords: -
 Abstract: A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates—the cleaved 5′ exon and the intron-3′ exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5′ splice site (i.e., the 3′ end of exon 1 and the 5′ end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2′-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5′ splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.

Details

show
hide
Language(s): eng - English
 Dates: 2012-05-242012-07
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1261/rna.033910.112
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: RNA
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 18 (7) Sequence Number: - Start / End Page: 1347 - 1357 Identifier: -