Blocky proline/glutamine patterns in the SFPQ intrinsically disordered region dictate paraspeckle formation as a distinct membraneless organelle
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Membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Many MLOs remain spatially compartmentalized, avoiding fusion or engulfment with one another. MLOs are formed by dynamic multivalent interactions among biomolecules, exemplified by proteins with intrinsically disordered regions (IDRs). However, the molecular principles behind how IDRs maintain MLO independence remain poorly understood. Here, we investigated the proline/glutamine (P/Q)-rich IDR of SFPQ, a protein identified as a key factor in segregating paraspeckles from nuclear speckles, to uncover the principles underlying this process. Paraspeckle segregation analyses, using MS2 tethering of SFPQ mutants onto NEAT1 long noncoding RNA, revealed that P/Q residues within the SFPQ IDR, whose enrichment is conserved from humans to zebrafish, play a crucial role in its segregation activity. In addition to the amino acid composition, the blocky patterns of P/Q residues are required for the segregation from nuclear speckles. Among human IDRs exhibiting PQ-block patterns, BRD4 IDR shows strong sequence similarity to the SFPQ IDR, and its IDR exhibits segregation activity at a level comparable to that of SFPQ. Furthermore, our molecular dynamics simulation suggests that the PQ-blocky patterns required for the paraspeckle segregation do not correlate with the IDR characteristics necessary for self-assembly. Thus, these data suggest that the PQ-blocky patterns in IDRs represent a previously uncharacterized property, which plays a role in maintaining the independence of MLOs, possibly through a mechanism distinct from the conventional phase separation-promoting function of IDRs.
Significance
Intrinsically disordered regions (IDRs) are amino acid sequences typically enriched in specific residues and lacking stable 3D structures. Multivalent interactions among IDRs often drive phase separation and the formation of membraneless organelles (MLOs). Paraspeckles are distinct MLOs that form near nuclear speckles, with SFPQ as a key component. The proline (P)- and glutamine (Q)-rich IDR of SFPQ is crucial for maintaining paraspeckle independence from nuclear speckles. We demonstrate that a blocky pattern of P/Q residues within the SFPQ IDR is critical for this independence. The BRD4 IDR exhibits a similar PQ-blocky pattern and function. Our findings highlight the crucial role of the PQ-blocky pattern in maintaining paraspeckle independence and offer new insights into IDRs in phase separation.
