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The Supraspliceosome - A Multi-Task Machine for Regulated Pre-mRNA Processing in the Cell Nucleus.

Shefer K, Sperling J, Sperling R - Comput Struct Biotechnol J (2014)

Bottom Line: The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs.Notably, changes in these regulatory processing activities are associated with human disease and cancer.These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Pre-mRNA splicing of Pol II transcripts is executed in the mammalian cell nucleus within a huge (21 MDa) and highly dynamic RNP machine - the supraspliceosome. It is composed of four splicing active native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. Supraspliceosomes harbor protein splicing factors and all the five-spliceosomal U snRNPs. Recent analysis of specific supraspliceosomes at defined splicing stages revealed that they harbor all five spliceosomal U snRNAs at all splicing stages. Supraspliceosomes harbor additional pre-mRNA processing components, such as the 5'-end and 3'-end processing components, and the RNA editing enzymes ADAR1 and ADAR2. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-EM. A unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in-silico studies, localizing the five U snRNPs mostly within its large subunit, and sheltering the active core components deep within the spliceosomal cavity. The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs. It also harbors a quality control mechanism termed suppression of splicing (SOS) that, under normal growth conditions, suppresses splicing at abundant intronic latent 5' splice sites in a reading frame-dependent fashion. Notably, changes in these regulatory processing activities are associated with human disease and cancer. These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.

No MeSH data available.


Related in: MedlinePlus

Suppression of splicing (SOS). (A) A scheme depicting the two scenarios that can account for lack of latent splicing under normal growth conditions, despite the abundance of latent 5′SS sequences in introns. As discussed, we have ruled out the 1st scenario [79–81,87]. Boxes, exons; narrow boxes, latent exons; lines, introns; red octagon, stop codon. (B, C) A schematic model for the quality control function of SOS. (B) Left scheme, splicing at the authentic 5′SS; right column, splicing at the latent 5′SS. (C) Splicing at the latent 5′SS after elimination of the in frame stop codon. Blue stripes, exons; black line, intron; green narrow stripe, latent exon; red octagon, stop codon; circles, U snRNPs; orange ellipse (UAC), hypothesized AUG-binding complex of initiator-tRNA; orange triangles, hypothesized triplet-binding proteins; red triangle, stop-codon-binding protein.
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f0020: Suppression of splicing (SOS). (A) A scheme depicting the two scenarios that can account for lack of latent splicing under normal growth conditions, despite the abundance of latent 5′SS sequences in introns. As discussed, we have ruled out the 1st scenario [79–81,87]. Boxes, exons; narrow boxes, latent exons; lines, introns; red octagon, stop codon. (B, C) A schematic model for the quality control function of SOS. (B) Left scheme, splicing at the authentic 5′SS; right column, splicing at the latent 5′SS. (C) Splicing at the latent 5′SS after elimination of the in frame stop codon. Blue stripes, exons; black line, intron; green narrow stripe, latent exon; red octagon, stop codon; circles, U snRNPs; orange ellipse (UAC), hypothesized AUG-binding complex of initiator-tRNA; orange triangles, hypothesized triplet-binding proteins; red triangle, stop-codon-binding protein.

Mentions: The presence of such a large number of latent 5′SS raises the question: why splicing at latent 5′SSs (latent splicing) has not been detected under normal growth conditions? In principle, two scenarios can account for this phenomenon (Fig. 4A): (i) Splicing at latent 5′SSs does occur, but the nonsense mRNAs thus formed are rapidly and efficiently degraded, by any RNA surveillance mechanism (e.g., NMD) [74–78] to a level below detection; and (ii) Splicing events at intronic 5′SSs that are preceded by at least one stop codon in frame with the upstream exon are suppressed. We have tested the first scenario in a substantial number of independent experiments: It was thus shown that SOS was not affected (i.e. latent splicing was not elicited in the tested gene transcripts) upon abrogation or bypassing the NMD pathway by a number of ways: Inhibition of translation [79–81] and, in particular, inhibition of the pioneer round of translation, which had been shown to be essential for NMD [82], by expressing a dominant negative mutant of the translation initiation factor eIF2-α, did not elicit latent splicing [81]. Furthermore, RNAi of the NMD genes hUpf1 and hUpf2, or the expression of three mutants of hUpf1 that abrogated NMD [83], did not elicit latent splicing [80]. Because Upf1 is essential for mammalian NMD [84–86], these data do not fit a model that could attribute the lack of latent splicing to a rapid and complete degradation of latent mRNAs by NMD. We also ruled out degradation of latent mRNA by a yet unknown RNA degradation mechanism by showing that constructs in which we forced formation of latent mRNA, through its expression from a plasmid harboring the already spliced DNA, express latent mRNA at levels only slightly lower than the level of authentic mRNA expressed from a plasmid harboring the already spliced DNA at the authentic 5′SS [87]. On the other hand, the data summarized above, fit the second scenario and invoke a mechanism termed suppression of splicing (SOS) that suppresses splicing involving latent alternative 5′SSs whose usage could introduce an intronic stop codon into the resultant mRNA [88].


The Supraspliceosome - A Multi-Task Machine for Regulated Pre-mRNA Processing in the Cell Nucleus.

Shefer K, Sperling J, Sperling R - Comput Struct Biotechnol J (2014)

Suppression of splicing (SOS). (A) A scheme depicting the two scenarios that can account for lack of latent splicing under normal growth conditions, despite the abundance of latent 5′SS sequences in introns. As discussed, we have ruled out the 1st scenario [79–81,87]. Boxes, exons; narrow boxes, latent exons; lines, introns; red octagon, stop codon. (B, C) A schematic model for the quality control function of SOS. (B) Left scheme, splicing at the authentic 5′SS; right column, splicing at the latent 5′SS. (C) Splicing at the latent 5′SS after elimination of the in frame stop codon. Blue stripes, exons; black line, intron; green narrow stripe, latent exon; red octagon, stop codon; circles, U snRNPs; orange ellipse (UAC), hypothesized AUG-binding complex of initiator-tRNA; orange triangles, hypothesized triplet-binding proteins; red triangle, stop-codon-binding protein.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232567&req=5

f0020: Suppression of splicing (SOS). (A) A scheme depicting the two scenarios that can account for lack of latent splicing under normal growth conditions, despite the abundance of latent 5′SS sequences in introns. As discussed, we have ruled out the 1st scenario [79–81,87]. Boxes, exons; narrow boxes, latent exons; lines, introns; red octagon, stop codon. (B, C) A schematic model for the quality control function of SOS. (B) Left scheme, splicing at the authentic 5′SS; right column, splicing at the latent 5′SS. (C) Splicing at the latent 5′SS after elimination of the in frame stop codon. Blue stripes, exons; black line, intron; green narrow stripe, latent exon; red octagon, stop codon; circles, U snRNPs; orange ellipse (UAC), hypothesized AUG-binding complex of initiator-tRNA; orange triangles, hypothesized triplet-binding proteins; red triangle, stop-codon-binding protein.
Mentions: The presence of such a large number of latent 5′SS raises the question: why splicing at latent 5′SSs (latent splicing) has not been detected under normal growth conditions? In principle, two scenarios can account for this phenomenon (Fig. 4A): (i) Splicing at latent 5′SSs does occur, but the nonsense mRNAs thus formed are rapidly and efficiently degraded, by any RNA surveillance mechanism (e.g., NMD) [74–78] to a level below detection; and (ii) Splicing events at intronic 5′SSs that are preceded by at least one stop codon in frame with the upstream exon are suppressed. We have tested the first scenario in a substantial number of independent experiments: It was thus shown that SOS was not affected (i.e. latent splicing was not elicited in the tested gene transcripts) upon abrogation or bypassing the NMD pathway by a number of ways: Inhibition of translation [79–81] and, in particular, inhibition of the pioneer round of translation, which had been shown to be essential for NMD [82], by expressing a dominant negative mutant of the translation initiation factor eIF2-α, did not elicit latent splicing [81]. Furthermore, RNAi of the NMD genes hUpf1 and hUpf2, or the expression of three mutants of hUpf1 that abrogated NMD [83], did not elicit latent splicing [80]. Because Upf1 is essential for mammalian NMD [84–86], these data do not fit a model that could attribute the lack of latent splicing to a rapid and complete degradation of latent mRNAs by NMD. We also ruled out degradation of latent mRNA by a yet unknown RNA degradation mechanism by showing that constructs in which we forced formation of latent mRNA, through its expression from a plasmid harboring the already spliced DNA, express latent mRNA at levels only slightly lower than the level of authentic mRNA expressed from a plasmid harboring the already spliced DNA at the authentic 5′SS [87]. On the other hand, the data summarized above, fit the second scenario and invoke a mechanism termed suppression of splicing (SOS) that suppresses splicing involving latent alternative 5′SSs whose usage could introduce an intronic stop codon into the resultant mRNA [88].

Bottom Line: The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs.Notably, changes in these regulatory processing activities are associated with human disease and cancer.These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Pre-mRNA splicing of Pol II transcripts is executed in the mammalian cell nucleus within a huge (21 MDa) and highly dynamic RNP machine - the supraspliceosome. It is composed of four splicing active native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. Supraspliceosomes harbor protein splicing factors and all the five-spliceosomal U snRNPs. Recent analysis of specific supraspliceosomes at defined splicing stages revealed that they harbor all five spliceosomal U snRNAs at all splicing stages. Supraspliceosomes harbor additional pre-mRNA processing components, such as the 5'-end and 3'-end processing components, and the RNA editing enzymes ADAR1 and ADAR2. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-EM. A unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in-silico studies, localizing the five U snRNPs mostly within its large subunit, and sheltering the active core components deep within the spliceosomal cavity. The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs. It also harbors a quality control mechanism termed suppression of splicing (SOS) that, under normal growth conditions, suppresses splicing at abundant intronic latent 5' splice sites in a reading frame-dependent fashion. Notably, changes in these regulatory processing activities are associated with human disease and cancer. These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.

No MeSH data available.


Related in: MedlinePlus