Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
In-Silico Study of the Arrangement of the snRNPs within the Native Spliceosome
The elaborate process of transforming the information coded in the DNA to protein molecules is performed by several large and intricate molecular machines: RNA polymerase II transcribes the coded genes to pre-mRNAs, the spliceosome processes the pre-mRNAs, eliminating noncoding introns and producing functional mRNAs, and the ribosome translates the genetic code embedded in the mRNAs and catalyzes the synthesis of proteins.
The spliceosome is a huge mega-Dalton ribonucleoprotein (RNP) assembly. Electron microscopy structures of the native spliceosome and of several spliceosomal subcomplexes, such as the spliceosomal U snRNPs, are available but the spatial arrangement of the latter within the native spliceosome is not known. We developed the fitEM2EM computational tools (Frankenstein et al, 2008), that match and dock low resolution structures. Next, we represented each spliceosomal subcomplex by an ensemble of normal-modes conformers and designed a new “conformer selection” procedure that efficiently fitted the thousands of conformers into the native spliceosome envelope.
Despite the low resolution limitations, we obtained only one model that complies with the available biochemical data. Our model localizes the five small nuclear RNPs (snRNPs) mostly within the large subunit of the native spliceosome, requiring only minor conformation changes. The remaining free volume presumably accommodates additional spliceosomal components. Moreover, the ample free volume suggests that structural modulations of the snRNPs can be tolerated while keeping the integrity of the spliceosome assembly. The constituents of the active core of the spliceosome are juxtaposed in our model, forming a continuous surface deep within the large spliceosomal cavity. This cavity emerges as the site of mRNA binding and splicing; its depth provides a sheltered environment for the splicing reaction (Frankenstein et al, 2012).
To experimentally localize U snRNPs within the native spliceosome and validate the model, we use gold nanoclusters of 1.5 nm in diameter, covalently attached to antisense oligodeoxynucleotides, each complementary to one of the spliceosomal U snRNAs.
This research has been supported by the US NIH, grant GM079549 to R.S. and J.S., and the Helen and Milton Kimmelman Center for Biomolecular Structure and Assembly at the Weizmann Institute of Science.
Z. Frankenstein, J. Sperling, R. Sperling and M. Eisenstein (2012). A Unique Spatial Arrangement of the snRNPs within the Native Spliceosome Emerges from In-Silico Studies. Structure, 20:1097-1106.
Ziv Frankenstein 1
Departments of Structural Biology1