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

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.

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

“…The gradient was subdivided into four parts: A-D. Fraction A contained proteins that sedimented faster than 200S and was devoid of spliceosomes; fraction B contained supraspliceosomes that sedimented in the 200S region (37,48,49), fraction C contained native spliceosomes that sedimented at 60-70S (37,41,50), and fraction D contained proteins and small RNAs that appeared as free proteins or assembled into small RNP complexes. SNORD2, SNORD60, and SNORD78 were present in fractions B and C associated with spliceosomes but devoid of fibrillarin (Figs.…”

Structural studies of the endogenous spliceosome – The supraspliceosome

“…Our approach for the study of the endogenous spliceosome was through analysis of complexes isolated from cell nuclei under physiological conditions and having intact RNA (reviewed in Refs [6, 31, 32]).…”

“…1). This 21 MDa RNP complex [30] is composed of four active native spliceosomes, each resembling the in vitro assembled spliceosome, which are connected via the pre-mRNA (reviewed in Refs [6, 31, 32]).…”

“…We have demonstrated that AS factors are predominantly found in supraspliceosomes, and that AS is regulated there [39, 40]. Notably, a quality control mechanism of pre-mRNA processing was identified in supraspliceosomes [31, 41, 42], as well as all the additional processing activities, such as 5’-end and 3’-end processing, and RNA editing [35, 43]. Furthermore, intronic pre-microRNAs were found in supraspliceosomes and their processing occurs there [44, 45].…”

“…Supraspliceosomes were also visualized in the cell nucleus from the site of transcription to the nuclear pore [48]. Thus, the supraspliceosome – the endogenous spliceosome - is functional in all nuclear processing activities that the pre-mRNA has to undergo [6, 31, 32]. …”