SART3-Dependent Accumulation of Incomplete Spliceosomal snRNPs in Cajal Bodies

Abstract

Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain a homeostatic balance of mature snRNPs in the nucleoplasm.

Coilin: The first 25 years

“…73 We recently showed that increased concentration of unassembled or defective snRNPs triggers formation of CBs in cells that are normally devoid of CBs. 37 This suggests that incomplete snRNPs enhance coilin self-interaction properties and induce formation of CBs through an unknown mechanism.…”

“…This finding suggests that coilin is part of a quality control mechanism that proofreads final snRNP assembly. 37 Again, this function might not be essential under normal conditions but becomes important when snRNP assembly is perturbed or when transcription and splicing rates are high, producing a large quantity of mono-snRNPs that require recycling and reassembly. Coilin-dependent concentration of mono-snRNPs in CBs would increase their assembly rate and at the same time prevent incomplete snRNPs from entering splicing reaction.…”

“…Black bars signify parts of coilin known to interact with indicated proteins/RNA. 12,14,15,33,37,48,58,[84][85][86][101][102][103][104] (B) The current hypothesis of nuclear body assembly suggests that protein and RNA components (colored shapes) tend to have more interactions (double arrows) among themselves as compared to other molecules (blue circles) in the environment. When the components reach a critical concentration locally, liquid phase separation occurs and creates the nuclear body.…”

“…83 snRNP-specific proteins are highly active in this assay for CB formation, which is consistent with a role for snRNPs in the context of naturally occurring CBs. 37,73 Coilin: A non-canonical RNA binding protein Despite the fact that coilin does not contain a canonical RNA binding motif, its X. laevis coilin was shown to interact with poly-U and poly-G in vitro. It was noted by the same authors that the N-terminal self-interaction domain may contain a degenerate RNA recognition motif.…”

Spectral imaging to visualize higher-order genomic organization

“…We have implemented this method, which we have named spectraFISH, for the study of 5 distinct genes or CTs with the CB, a major NB involved in spliceosomal snRNP biogenesis [31][32][33] and found frequently in cancer cells. 21 This nuclear structure actively coordinates 34 a network of gene pairing interactions with major and minor spliceosome U snRNA genes, [12][13][14]35 and other highly active intron-encoded U snoRNA/scaRNA genes, 21,36 but also histone gene clusters (e.g.…”