TY - JOUR
T1 - Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine
AU - Medalia, Ohad
AU - Typke, Dieter
AU - Hegerl, Reiner
AU - Angenitzki, Mina
AU - Sperling, Joseph
AU - Sperling, Ruth
PY - 2002
Y1 - 2002
N2 - Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.
AB - Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.
KW - Charged lipid monolayer
KW - Cryoelectron microscopy
KW - Cryoelectron tomography
KW - Image restoration
KW - lnRNP complexes
KW - Pre-mRNA processing
KW - Three-dimensional image reconstruction
UR - http://www.scopus.com/inward/record.url?scp=0036422206&partnerID=8YFLogxK
U2 - 10.1016/S1047-8477(02)00027-8
DO - 10.1016/S1047-8477(02)00027-8
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C2 - 12160703
AN - SCOPUS:0036422206
SN - 1047-8477
VL - 138
SP - 74
EP - 84
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1-2
ER -