The KH domains of hnRNP K bind both DNA and RNA, and several studies have used DNA instead of RNA in their experiments 21, 22. The hnRNP K-SIRLOIN affinity obtained with DNA in our study was highly similar to a recent reported affinity with RNA (1.1 µM vs. 0.7 µM) 23. It has been shown previously that KH1 does not bind to CTRs 23. We also determined that KH1 does not bind to DNA when purified as a single domain (Table 1). However, when properly folded with the nearby KH2 domain, KH1 was active in DNA binding similar to the other KH domains. Thus, proper protein purification/folding should be carefully verified when designing or using protein mutants.
Most reports have considered hnRNP K as a poly-C binding protein 23, 24. A previous analysis proposed that KH3 binds only to TCCC or CCCC 21. Our structural and biochemical studies showed that KH3 could bind to a CCTC motif, suggesting that the third position does not need to be a C. For the fourth position, given its high degree of flexibility (Fig. S5), substituting it with a T or U would probably not reduce KH3 binding affinity. Thus, we updated the definition of KH3 recognition (which may also work for KH1 and KH2), i.e., except the second base that is exclusively C 21, the other three bases could be any pyrimidine (C, T, or U).
This new definition can explain the partial conservation of T/U in hnRNP K-bound sequences, particularly that some of the hnRNP K binding sequences lack poly C patches 23, 24. Furthermore, this definition predicts that the CTR2 of SIRLOIN may bind to KH domains by two overlapping motifs, being either TCTC or TCCT (CTR2 contains no TCCC or CCCC motif). In solution, it is possible that due to the presence of DNA/RNA secondary structure 23 or steric hindrance only one of the motifs is selectively bound by a KH domain, as in the crystal structure that only CCTC in CTR3 is bound by KH3 (Fig. 2C). It has been previously shown that two tandem unspaced CTR motifs allow simultaneous binding to two KH domains 21. The fourth identified CTR in SIRLOIN, a CCTC motif, is separated from CTR2 and CTR3 by one and two nucleotides, respectively, rendering SIRLOIN capable to bind four KH domains simultaneously. Together with the crystal structure, ITC, EMSA, and size exclusion results (Fig. 2C, 2F, and 5B, Table 1), it is concluded that SIRLOIN is a tetravalent KH domain binding sequence, instead of a trivalent.
The ITC analysis showed that increasing the number of CTRs increased the binding affinity of each KH domain. This agrees with the avidity theory whereby tethering multiple binding sites creates ‘forced proximity’ 25. However, increasing the number of KH domains did not increase SIRLOIN binding affinity (Table 1). Similarly, deleting KH3 did not abruptly reduce its affinity for different CTRs 23. It has been proposed that the protein hnRNP K functions in chromatin remodeling, and that tandem KH domains in a protein can remodel the RNA structure 26, 27. Thus, the binding energy gained through tethering multiple KH domains may be counter-balanced by the energy spent on remodeling the DNA/RNA secondary structure (to a higher energy state). In agreement with this speculation, C-rich RNAs without secondary structures have a higher affinity for hnRNP K 23.
Previous reports have shown that hnRNP K contains an NLS and a KNS domain which facilitate its nuclear import 11, 27. Here, deleting NLS, but not KNS, partially inhibited the nuclear import of hnRNP K. However, our results do not exclude the function of KNS in nuclear import. The finding that the NLS deletion mutant was also mainly nuclear localized suggests that nuclear import of hnRNP K is redundant, similar to nuclear import of many other proteins, such as FUS 28, 29, 30. In particular, hnRNP K contains several RG/GR motifs that may be recognized by transportin 1 31, 32. It remains unknown whether other hnRNP K import pathways can simultaneously import SIRLOIN into the nucleus.
Lubelsky reported that SIRLOIN promotes nuclear accumulation of a mRNA when fused to that mRNA 7. Here, it was demonstrated that SIRLOIN, but not its CTR mutant, entered the cell nucleus when directly added into cells (Fig. 1E). Tethering two SIRLOIN repeats in tandem promoted this process, in line with the report that RNAs containing more SIRLOIN repeats are more nuclear-localized than those with one SIRLOIN 7. Therefore, besides nuclear retention 33, nuclear import may be another mechanism of nuclear localization for SIRLOIN-containing DNAs/RNAs. Landerer and his colleague found that mitochondrial-encoded lncRNAs could be exported from mitochondria and relocalized into the nucleus 34. Thus, there exists a lncRNA nuclear import mechanism in cells, and NIRs and hnRNP K may play a role in this step.
A common feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the abrupt formation of SGs or cytoplasmic inclusions by different RBPs including FUS, TDP-43, and hnRNP A1 17, 35. These RNA-binding proteins and hnRNP K have similar characteristics, e.g., all contain RNA-binding domains (also called RNA recognition motifs) and R/G rich disordered domains, but little is known about mislocalization of hnRNP K in the cytoplasm of cells with neuronal disease 36. Here, it was found hnRNP K had a high tendency to enter SGs, which was independent of DNA binding but partially dependent on the classical NIRs (Fig. 8). A previous report suggested that SG depletion of hnRNP K prevents SG accumulation of TDP-43, a binding partner of hnRNP K 15. Whether SG localization of hnRNP K plays a role in health and disease warrants further studies.
One of the essential proteins of the nuclear-localized membrane-less organelle paraspeckles is hnRNP K 37. The mechanism of hnRNP K SG localization may share some features with its localization in paraspeckles 38. The RGG domain has been proposed to non-specifically bind DNA/RNA, and promote its SG (and possibly paraspeckle) localization by interacting with other RNAs or proteins in SGs 39. Even without the RGG domain, hnRNP K formed a oligomeric complex with SIRLOIN in the context of size exclusion. The affinities of all CTR-KH domain pair in this complex are in the middle micromolar range (Table 1). Thus, it may not be a specifically configured complex, but rather a complex with dynamic and heterogeneous configurations, featuring a weak multivalent interaction that is often observed in membrane-less organelles 19, 20. At higher concentrations of hnRNP K and SIRLOIN, particularly in SGs, hnRNP K and SIRLOIN may not form such a 4:3 complex, but rather a more complex network involving other CTRs and KH domains in SGs (beyond the interaction complexity of RGG-mediated networks).
The recruitment of hnRNP K into SGs co-deposited bound SIRLOIN into SGs. This is analogous to storing mRNAs in SGs by RBPs under cellular stress 13. The nuclear import and SG deposition phenomenon is probably not limited to SIRLOIN but also applicable to other hnRNP K binding motifs 23. A previous study suggested that P body (another cytoplasmic membrane-less organelle) association of lncRNA may affect mRNA translation by regulating the types of mRNA associated with P bodies 40, but the function of lncRNA SG localization is unknown. It remains to be explored how the nuclear and SG localization of SIRLOIN is regulated, but the NIR concentration may be a factor (Fig. 8).
Several importins are demonstrated to prevent RBPs from accumulating in SGs 30, 32, 35. Likewise, our results show that SG association of hnRNP K was reduced by a high concentration of classical NIRs (Fig. 8). However, a low concentration of NIRs enhanced its SG localization. The NIRs are able to equilibrate in and out of the membrane-less organelles including SGs 41. It is reasoned that a low concentration of NIR promoted SG accumulation of hnRNP K via enhancing its SG accessibility, in addition to preventing its non-specific cytoplasmic staining (Fig. 6A). Unlike hnRNP K, SG accumulation of SIRLOIN was more dependent on NIRs. This warrants further investigation to understand why hnRNP K alone is inefficient at importing SIRLOIN into SGs.