Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa
Basic protein information
Accession P26368
Common name U2AF65
Gene U2AF2
Organism Homo sapiens
Uniprot name Splicing factor U2AF 65 kDa subunit
Basic LLPS information
Organelle nuclear speckle
Type of experimental evidence
Protein region(s) mediating LLPS
27
-
62
RS domain
149
-
337
RRMs
385
-
466
UHM domain
Based on the experimental results of the following publication: 31271494
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
In cells, knockdown of either U2AF65 or CAPERa improves the inclusion of cassette exons that are preceded by repeated pyrimidine-rich motifs. In the second step of spliceosome assembly, SF1 that was bound to the branchpoint sequence is replaced by the U2 snRNA-containing ribonucleoprotein (U2 snRNP) with the help of U2AF65. The N-terminal arginine- and serine-rich (RS) domain of U2AF65 contacts the branchpoint sequence (BPS) and favors the formation of a U2 snRNA-BPS duplex. The U2AF65 UHM domain engages interactions with ULM motifs of the U2 snRNP subunit SF3b155. The results support a model in which liquid-like assemblies of U2AF65 and CAPERa on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. RNA increases RS-dependent sedimentation most probably by favoring local concentration of U2AF65 on repeated binding sites for its RRMs. The U2AF65 RS domain mediates multivalent interactions in vitro and localization to compartments thought to originate from LLPS in vivo. At the light of the in vitro and in cells results, a mechanistic model is proposed in which the recruitment of U2 snRNP at the 3' intronic sequences is regulated by liquid-like assemblies of U2AF65 and CAPERa generated by self-attracting RS domains, multiple UHM–ULM interactions with SF3B155, and bindings of RRMs to repeated pyrimidine-rich sequences. In all, U2AF65 assemblies contribute to sequence-specific splice site recognition (PMID: 31271494).
Literature supporting the LLPS: 31271494
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor
Binding partners (at biological protein concentrations)
1) SF3b155c (promotes LLPS through its ULM motifs binding to the UHM domain of U2AF65) 2) SPY-rich RNAs (promote LLPS through binding to the RRMs of U2AF65)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
SPY-rich RNAs (intronic RNA with repeated pyrimidine tracts)
Molecular interaction types contributing to LLPS
multivalent domain-motif interactions (PMID: 31271494) protein-RNA interaction (PMID: 31271494)
Determinants of phase separation and droplet properties
1) protein concentration of U2AF65 2) salt concentration 3) crowding agent concentration
Membrane cluster No
Partner-dependent No
RNA-dependent No
PTM required No
Domain-motif interactions Yes
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
Phase-contrast microscopy revealed the presence of droplets of U2AF65 which were sensitive to salt, SDS, concentration, and PEG-induced crowding as expected for a LLPS mechanism. This LLPS mechanism was also supported by the dynamics of these objects as evidenced by time-lapse microscopy. To ascertain the role of the low complexity RS domain in LLPS of U2AF65, the RS domain fused to GST was purified. The results indicate that liquid droplets are formed with the RS domain alone. In addition, deletion of the RS domain resulted in reduced sedimentation of U2AF65, indicating that this domain is mostly responsible for the formation of U2AF65 assemblies. SPY-rich RNAs had a concentration-dependent positive effect on the formation of U2AF65 assemblies. The formation of U2AF65 assemblies upon DNM2 RNA addition was exacerbated by the presence of the SF3b155 multi-ULM domain but not by a mutated domain lacking the essential tryptophan residues of the seven ULM motifs (tryptophan residues replaced by alanine). While SF3b155c was mainly soluble even at high concentration, it was efficiently recruited to U2AF65 assemblies in the presence of SPY-rich but not SPY-poor RNA. This effect of SF3b155c on U2AF65 assemblies was not observed for SF1c which presents a single ULM. Similar to repeated SPY sequences on RNA, the multi-ULM domain of SF3b155 probably increases the local concentration of U2AF65, thereby favoring liquid-like U2AF65 assemblies. In vivo overexpressed myc-tagged U2AF65 presented a distribution (protein localization) similar to the endogenous protein, but deletion of the RS domain dramatically reduced its sedimentation as confirmed by immunofluorescence microscopy. U2AF65 showed a co-localization with SF3b155 (PMID: 31271494).
Experimental observations supporting the liquid material state of the condensate
morphological traits (PMID: 31271494)