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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

Structure of the native spliceosome.(A, B) Two different views of the structure of the native spliceosome reconstructed at 20 Å resolution from cryo-images [31]. (C) The high-density mass region of the native spliceosome (red) represents the stable RNAs within the structure of the native spliceosome. The large subunit of the native spliceosome is thus a suitable candidate to harbor the five-spliceosomal U snRNPs. Adapted from Azubel et al. [31]. (D) A unique spatial arrangement of the U snRNPs within the native spliceosome emerges from in-silico studies [32]. The native spliceosome is transparent; U4/U6.U5 tri-snRNP is colored by functional regions, with U5 snRNP in pink and the region attributed to loop I in black [93]; U4/U6 in beige-orange; U2 snRNP subcomponent SF3b is in green; and U1 snRNP is blue. Adapted from Frankenstein et al. [32].
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f0010: Structure of the native spliceosome.(A, B) Two different views of the structure of the native spliceosome reconstructed at 20 Å resolution from cryo-images [31]. (C) The high-density mass region of the native spliceosome (red) represents the stable RNAs within the structure of the native spliceosome. The large subunit of the native spliceosome is thus a suitable candidate to harbor the five-spliceosomal U snRNPs. Adapted from Azubel et al. [31]. (D) A unique spatial arrangement of the U snRNPs within the native spliceosome emerges from in-silico studies [32]. The native spliceosome is transparent; U4/U6.U5 tri-snRNP is colored by functional regions, with U5 snRNP in pink and the region attributed to loop I in black [93]; U4/U6 in beige-orange; U2 snRNP subcomponent SF3b is in green; and U1 snRNP is blue. Adapted from Frankenstein et al. [32].

Mentions: The four substructures of the supraspliceosome are interconnected in a flexible way and may thus adopt different angular settings, which impose a significant restriction on reaching high resolution in EM image analyses. We have therefore developed a methodology to isolate the native spliceosomes from supraspliceosomes, by specific cleavage of the general population of pre-mRNAs, while keeping the snRNAs within these substructures intact [19]. The 3-D structure of the native spliceosome was determined by the cryo-EM single particle technique at a resolution of 20 Å, and revealed an elongated globular particle composed of a large and a small subunit (Fig. 2A–C) [31]. The two subunits are interconnected to each other leaving a tunnel in between, which is large enough to allow the pre-mRNA to pass through (Fig. 2A). The other side of the native spliceosome exposes a cavity that could provide a place to transiently store the labile pre-mRNA and protect the part of the pre-mRNA that is not directly involved in the splicing reaction from non-specific degradation (Fig. 2B). Because RNA is denser than protein, high-density regions can provide some information about its internal localization. The large subunit was thus proposed as a suitable candidate to accommodate the five spliceosomal U snRNPs, as the high density regions were found there (Fig. 2C) [31].


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)

Structure of the native spliceosome.(A, B) Two different views of the structure of the native spliceosome reconstructed at 20 Å resolution from cryo-images [31]. (C) The high-density mass region of the native spliceosome (red) represents the stable RNAs within the structure of the native spliceosome. The large subunit of the native spliceosome is thus a suitable candidate to harbor the five-spliceosomal U snRNPs. Adapted from Azubel et al. [31]. (D) A unique spatial arrangement of the U snRNPs within the native spliceosome emerges from in-silico studies [32]. The native spliceosome is transparent; U4/U6.U5 tri-snRNP is colored by functional regions, with U5 snRNP in pink and the region attributed to loop I in black [93]; U4/U6 in beige-orange; U2 snRNP subcomponent SF3b is in green; and U1 snRNP is blue. Adapted from Frankenstein et al. [32].
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232567&req=5

f0010: Structure of the native spliceosome.(A, B) Two different views of the structure of the native spliceosome reconstructed at 20 Å resolution from cryo-images [31]. (C) The high-density mass region of the native spliceosome (red) represents the stable RNAs within the structure of the native spliceosome. The large subunit of the native spliceosome is thus a suitable candidate to harbor the five-spliceosomal U snRNPs. Adapted from Azubel et al. [31]. (D) A unique spatial arrangement of the U snRNPs within the native spliceosome emerges from in-silico studies [32]. The native spliceosome is transparent; U4/U6.U5 tri-snRNP is colored by functional regions, with U5 snRNP in pink and the region attributed to loop I in black [93]; U4/U6 in beige-orange; U2 snRNP subcomponent SF3b is in green; and U1 snRNP is blue. Adapted from Frankenstein et al. [32].
Mentions: The four substructures of the supraspliceosome are interconnected in a flexible way and may thus adopt different angular settings, which impose a significant restriction on reaching high resolution in EM image analyses. We have therefore developed a methodology to isolate the native spliceosomes from supraspliceosomes, by specific cleavage of the general population of pre-mRNAs, while keeping the snRNAs within these substructures intact [19]. The 3-D structure of the native spliceosome was determined by the cryo-EM single particle technique at a resolution of 20 Å, and revealed an elongated globular particle composed of a large and a small subunit (Fig. 2A–C) [31]. The two subunits are interconnected to each other leaving a tunnel in between, which is large enough to allow the pre-mRNA to pass through (Fig. 2A). The other side of the native spliceosome exposes a cavity that could provide a place to transiently store the labile pre-mRNA and protect the part of the pre-mRNA that is not directly involved in the splicing reaction from non-specific degradation (Fig. 2B). Because RNA is denser than protein, high-density regions can provide some information about its internal localization. The large subunit was thus proposed as a suitable candidate to accommodate the five spliceosomal U snRNPs, as the high density regions were found there (Fig. 2C) [31].

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