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These findings highlight the important role of large, pre-formed, complexes in pre-mRNA splicing in vivo. It has also been pointed out that such intermediate states in spliceosome assembly in vitro may not occur in vivo [ 72 , 73 ]. Mass spectrometry analysis of the affinity purified supraspliceosomes revealed an average of protein groups, higher amount than the protein groups detected previously for the analysis of a general population of supraspliceosomes purified from HeLa cells [ 74 ].
This difference likely reflects the difference in purification methods and instrumentation used. The SR proteins, which play a role in alternative and constitutive RNA splicing [ 76 ], were previously shown to be part of the supraspliceosome [ 28 , 39 , 40 , 52 ].
The proteomic analyses of supraspliceosomes assembled on AdML-WT and AdML-Mut transcripts strengthen the above conclusion that the supraspliceosome is the pre-mRNA processing machine that is pre-assembled throughout all splicing stages, as the core components of the spliceosome can be seen in all these stages.
The analyses also show the dynamic nature of the supraspliceosome during the splicing stages, as different hnRNP isoforms were found in supraspliceosomes from the different steps of splicing [ 38 ]. We have shown that oligonucleotide-directed RNase H digestion of the pre-mRNA within supraspliceosomes gave rise to native spliceosomes, yet, native spliceosomes could not be reconstituted into supraspliceosomes just by the addition of magnesium cations [ 29 ].
These experiments demonstrated that the pre-mRNA connects the native spliceosomes within the supraspliceosome, and highlighted the essential role of the pre-mRNA for the assembly of the supraspliceosome.
watch We confirmed the conclusion that the pre-mRNA is required for supraspliceosome assembly, by showing that supraspliceosomes can be reconstituted by back-addition of pre-mRNA. Incubation of purified native spliceosomes with in vitro transcribed pre-mRNAs having an intron flanked by two exons, followed by TEM visualization of the negatively stained complexes revealed that native spliceosomes were thus reconstituted into tetrameric supraspliceosomes [ 37 ] see also Section 5.
The maturation of the precursors of mRNA to a functional transcript in eukaryotic cells is a complex, multistep process. It includes the formation of RNP. RNP Particles, Splicing and Autoimmune Diseases (Springer Lab Manuals): Medicine & Health Science Books @ prethanddownmarrent.gq
The reconstituted supraspliceosomes, obtained from each of three tested pre-mRNAs, are composed of four native spliceosomes and are similar to supraspliceosomes isolated from living cells with respect to their morphology and dimensions. Control experiments showed that no supraspliceosomes were reconstituted upon addition of double or single stranded DNA encoding for the respective pre-mRNAs. These experiments highlighted the essential role of the pre-mRNA for the integrity of the supraspliceosome [ 37 ].
A prerequisite for efficient and precise alternative splicing and pre-mRNA processing within the supraspliceosome is that each complex should process a single RNA transcript at a time, and this hypothesis has been implied in the supraspliceosome model [ 6 , 31 , 32 , 37 ].
To test this hypothesis, we used our above procedure for reconstituting tetrameric supraspliceosomes from monomeric native spliceosomes [ 37 ], this time, with an exogenously added gold-tagged synthetic pre-mRNA [ 40 ]. For this experiment Fig. EM visualization Fig. These experiments confirm that each supraspliceosome is assembled on one pre-mRNA [ 40 ]. Left panel, staining with ethidium bromide; right panel, staining of the blotted gel by silver enhancement.
White arrowheads point to gold-enhanced Nanogold. Bar represents 50 nm. Adapted from Ref [ 40 ].
Structural analysis of native spliceosomes in the context of intact supraspliceosomes, using electron microscopy combined with image processing revealed good correlation between the structure of the isolated native spliceosome, solved by cryo-EM, and the native spliceosome within the intact supraspliceosome [ 80 ]. Furthermore, this study enabled us to study the arrangement of the native spliceosomes within the intact particle Fig.
The edges of the small subunits, which are in the center of the supraspliceosome, form a right angle and thus facilitate close contacts between the small subunits generating a four-fold pattern. This pattern is observed in individual and averaged images [ 80 ]. Intact supraspliceosomes were classified by Correspondence Analysis and Hierarchical Ascendant Classification [ 80 ].
One of the classes is depicted showing the close contact between neighboring native spliceosomes in the center of the supraspliceosome, where the small subunits of the native spliceosomes are located.
The contacts between the neighboring small subunits form a right-angled cross that reflects a four-fold arrangement. Scale bars represent 10 nm. Adapted from Ref [ 80 ]. The supraspliceosome is a stand-alone macromolecular machine Fig. The small subunit of each native spliceosome is placed at the center of the supraspliceosome [ 80 ], thus allowing communication between the native spliceosomes, which is a crucial element for regulated alternative splicing and for quality control of the resulting mRNAs. This setting places the large subunit of each native spliceosome, where catalysis by the U snRNPs presumably takes place, in the periphery of the supraspliceosome.
This setting was supported by the in-silico study showing unique localization of the U snRNPs within the native spliceosome [ 69 ]. The supraspliceosome structure provides a platform to juxtapose exons about to be spliced, and each of the four native spliceosomes, resembling an in vitro assembled spliceosome, can splice the intron wound around it Fig. The supraspliceosome is a multiprocessor machine that can simultaneously splice four introns — not necessarily in a consecutive manner.
This configuration enables examination, prior to introns excision, if correct splice junctions will be combined, and allows rearrangement of splice junction combinations to select the appropriate ones, thus ensuring the fidelity of splicing and alternative splicing. The rotated volumes fitted well into the parts of neighboring sub-complexes seen in the average image [ 80 ]. Schematic models of the supraspliceosome in which the pre-mRNA introns in blue, exons in red is connecting four native spliceosomes [ 29 , 37 , 80 ].
The supraspliceosome presents a platform onto which the exons can be aligned and splice junctions can be checked before splicing occurs. A The pre-mRNA that is not being processed is folded and protected within the cavities of the native spliceosome. B When a staining protocol that allows visualization of nucleic acids was used, RNA strands and loops were seen emanating from the supraspliceosomes [ 30 ]. Under these conditions, the RNA kept in the cavity likely unfolded and looped-out. In the looped-out scheme an alternative exon is depicted in the upper left corner.
The supraspliceosome model predicts that each transcript will be assembled in a tetrameric supraspliceosome. After processing of four introns the RNA roles in to place a new subset of introns for processing. At the other hand, transcripts with less than four introns are also assembled in supraspliceosomes [ 37 , 38 ]. The interactions of the RNA with the native spliceosomes are presumably sufficient to hold the structure together.
The characterization of the supraspliceosome was performed on the nucleoplasmic complex. Thus, information on the initial stages of its assembly in vivo , whether it occurred in a step-wise manner, or it involved pre-assembled components is yet lacking. However, it is established, that unlike the in vitro spliceosome, all five-spliceosomal U snRNPs are associated with the nucleoplasmic supraspliceosome at all stages of splicing. The unique spatial arrangement of the U snRNPs within the native spliceosome that emerged from our in-silico studies [ 69 ], also revealed that there is ample free volume left within the native spliceosome after localization of the spliceosomal U snRNPs, indicating that structural modulations of the U snRNPs during the steps of the splicing reaction can be tolerated while keeping the integrity of the spliceosome assembly.
This is consistent with the recent high-resolution structures of spliceosome complexes by cryo-EM revealing dynamic changes between the spliceosome complexes.
Notably, movement of the pre-mRNA within the supraspliceosome, proposed in our model is consistent with movement of pre-mRNA elements observed between the different spliceosome subcomplexes [ 11 — 17 , 19 ]. The supraspliceosome model differs from the model derived from in vitro studies that predicts the assembly of a spliceosome on each intron [ 81 ].
It is however consistent with the cotranscriptional splicing model and especially with the model of cotranscriptional commitment to splicing [ 23 , 82 ]. The supraspliceosome provides a unique and general machine that encompasses the extensive network of interactions and offers coordination and regulation of the different splicing events that a multiintronic pre-mRNA has to undergo.
Thus, supraspliceosomes harbor components of all pre-mRNA processing activities, thus representing the nuclear pre-mRNA processing machine. Although the pattern and kinetics of the processing activities will likely be determined for each individual transcript, according to sequences of cis elements of that pre-mRNA, and the various factors that interact with these elements, the supraspliceosome is a general machine, capable of performing and regulating all the pre-mRNA processing activities that the pre-mRNA has to undergo before it can exit from the nucleus to the cytoplasm.
The recent advancements in cryo-EM, enabling structure determination at subnanometric resolution [ 7 ] have revolutionized the field of structural molecular biology, including the splicing field [ 8 — 19 ].. In view of this revolution in cryo-EM, further high-resolution imaging studies of the endogenous spliceosome are required. We Take advantage of the recent revolution in the field of structural analysis by cryo-EM, and of our advance in analyzing specific supraspliceosomes at defined functional states and alternatively spliced supraspliceosomes and native spliceosomes, for the cryo-EM structural analysis of supraspliceosomes and native spliceosomes using both cryo-ET and single particle technique as well as subtomograms averaging and in silico segmentations, in order to decipher the mechanism of regulation of alternative splicing, and how is the network of processing activities within the endogenous spliceosome coordinated.
We thank Aviva Petcho for excellent technical assistance and Minna Angenitski for help with the electron microscopy, and members of the Sperling labs for their contributions. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.
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See other articles in PMC that cite the published article. Abstract Pre-mRNA splicing is executed in mammalian cell nuclei within a huge 21 MDa and highly dynamic molecular machine — the supraspliceosome - that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature.