Peer review of: INTS10–INTS13–INTS14 form a functional module of Integrator that binds nucleic acids a (2020)

Editorial Note: This manuscript has been previously reviewed at another journal that is not operating 
a transparent peer review scheme. This document only contains reviewer comments and rebuttal 
letters for versions considered at Nature Communications.

REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

In this revised manuscript, Sabath and colleagues have refined their analysis on a newly identified 
structural/functional module of the Integrator complex. 

In its original version, this manuscript presented compelling biochemical and structural evidence of the 
INTS13/14 heterodimer. However, the data proposing INTS10 as a third component of the module 
were lacking some in vivo validation (which is now offered in the form of a native nuclear extract gel 
fractionation). 

In the second part of their manuscript, the authors identify an intriguing similarity between INTS13/14 
and Ku70/80, which propels their study of nucleic acid binding abilities of their module. The data 
presented suggest affinity for RNA, both single stranded and in the form of stem loops. The authors 
then propose a role for RNA binding in that it would help directing the cleavage module at the proper 
cutting site for nascent UsnRNAs. This hypothesis is further supported by data suggesting that the C 
terminal domain of INTS13 bridges the interaction with the 4-9-11 catalytic module. The in vivo data 
on the actual contribution of INTS13, INTS14 or INTS10 to UsnRNA processing (probing both 
endogenous loci and reporter constructs) partly supports their hypothesis, however the phenotype is 
somewhat modest and points to an overall ancillary role in UsnRNA biogenesis. 

In this improved version, the authors explore additional roles of Integrator and the potential 
contribution of the INTS13/14 module. They present data obtained with an HIV-promoter reporter 
system as a proxy to probe Pol2 pausing. Depletion of the module components results in a significant 
but moderate effect on pausing/elongation. Overall, these data still suggest that the main function of 
the module could lie elsewhere. 
Nonetheless, I think the overall results are interesting and provide significant advance to our 
understanding of this evolutionarily conserved nuclear complex. 

MINOR POINTS 
In the discussion, the authors point out, rightfully, that their findings about the cleavage module may 
hold implications for how INTS13 regulates myeloid differentiation. They further speculate that 
INTS13/14 and INTS11 might not be that functionally separated after all and that could be an 
antibody bias. While it is true that antibodies may preferentially recognize particular isoforms of a 
protein or even interfere with protein-protein interactions, data from that study (Mol Cell 2018) 
indicate that INTS13 antibodies do co-precipitate copious amounts of INTS11/9/4 when used on a 
total nuclear extract. Why would that be different in a fractionated extract? 

Reviewer #2 (Remarks to the Author): 

The authors have investigated the structures and interactions between subunits 10, 13 and 14 of the 
Integrator Complex (INTS10, 13 and 14). 
They have shown that INTS13 and INTS14 co-immunoprecipitate from HEK293 and insect cells after 
ectopic expression of tagged proteins as a stochiometric and stable heterodimer. They also carried out 
size exclusion chromatography, which confirms that a complex of INTS10/13/14 exists in nuclear 
extract. 
The crystal structure of INTS13/14 solved at 2.5 A indicates that although the two proteins do not 
have a high degree of primary sequence similarity, they have similar domain structures and form an 
intertwined pseudo-symmetric heterodimer. Sequence conservation suggests that this structure is 
conserved in all metazoans that express INTS13 and INTS14. In addition, the interlinked structure 
suggests that folding and dimerization are linked. 
Crosslinking mass spectrometry confirms that the conformation from crystallography is present in 
solution and shows that the complex oligomerizes. In addition, mutation of predicted interaction 
regions disrupt complex formation. 

The authors have also shown that INTS13/14 co-immunoprecipitates with INTS10 and that this 
heterotrimer is stable through purification. Mutational analysis indicates that INTS10 interacts with 
INTS14, in line with previous yeast 2-hybrid analyses of Drosophila INTS10 and INTS14 homologues. 
The authors define the VWA domain of INTS14 as necessary and sufficient for interaction with INTS10. 

Together this data defines INTS10/13/14 as a subcomplex of Integrator. 
As INTS13/14 has some homology to Ku70/Ku80, which binds nucleic acids, the authors have 
investigated the ability of INTS13/14 and INTS10/13/14 to interact with nucleic acid using EMSA and 
fluorescence polarization. INTS13/14 binds ssRNA best, whereas INTS10/13/14 has higher affinity for 
nucleic acid and prefers dsRNA. 

Both INTS13/14 and INTS10/13/14 co-immunoprecipitate INTS4/9/11, which they term the cleavage 
module and the authors present evidence that interaction is through the C-term of INTS13 and that 
the full INTS4/9/11 module is needed for interaction. 

Finally, the authors have assayed the effect of knockdown (KD) of INTS subunits on expression of 
renilla luciferase (rLuc) downstream from a U7 3’ box, with the rationale that cleavage at the 3’ box 
will down regulate rLuc expression. In line with this, KD of INTS11 causes a massive increase in rLuc 
expression. KD of INTS10, 13 or 14 has a more modest effect. In addition, deletion of the C-term of 
INTS13 appears to reduce the ability of INTS13 to complement INTS13 KD. In a very nice HIV-1 TAR-
mediated termination assay that the authors have set up, KD of INTS10 or 13 or 14 causes a modest 
increase in expression of a downstream gene, presumably due to increased readthrough. 

The structural studies are very well done and the structure for INTS13/14 is new and interesting. In 
addition, the nucleic acid binding studies support the idea that INTS10/13/14 could help Integrator 
interact with RNA, although there does not seem to be that much specificity. 

Unfortunately, the study doesn’t really succeed in going much beyond the structures and interactions 
to shed light on the molecular functions of the subunits. In addition, I think I would only conclude 
from the experiments in Figure 6b-g that the function of INTS10, 13 or 14 is not very important for 3’ 
processing and cleavage as the effect is so much less that INTS11 KD. Knockdown of INTS7, another 
non-catalytic subunit, has a much bigger effect in this assay. 

The evidence that the RNA-binding module recruits the cleavage module to 3’ boxes is therefore not 
strong as loss of this module has only a modest effect on cleavage of snRNA gene transcripts. The 
authors do note that KD of INTS10 or 13 or 14 has a relatively stronger effect in their HIV-1 
premature termination system. However, also in the HIV-1 assay, KD of INTS10, 13 or 14 has much 
less effect than KD of INTS7, suggesting that these subunits are playing a minor role in both systems 
or that KD is not getting rid of these subunits from the stably-associated complexes or their role in the 
tested systems just isn’t terribly important in cell lines. It is interesting that KD of INTS11 also doesn’t 
have a big effect in this assay. As KD of INTS11 (and INTS9) also has less of an effect than KD of 
INTS7 and 8 in the Drosophila MtnA system, perhaps cleavage is not the main mechanism inhibiting 
transcription in these systems as suggested by the authors. Relevant to this, KD of INTS10 or 13 or 
14 has very little effect in the Drosophila MtnA system. Perhaps an unbiased analysis of the effect of 
KD of these subunits on transcription and RNA processing or a more profound knockdown using 
degrons or a more in vivo system would help to determine whether these subunits play a major role in 
gene expression. 

Other comments 

Lines 10-12. The authors should make it clear that the studies on Integrator causing termination of 
paused RNAPII that they cite were in Drosophila. 

Figure 3a. Is INTS11 modified in fractions 16/18, where the authors say the INTS10/13/14 complex 
is? Looks to me that there are many subcomplexes eg INTS1/4/13/14 and 
INTS10/11(modified)/13/14. Also, INTS7 looks like it is running faster in fractions 16/18. 

Figure 7a. Should there really be scissors on the diagram? KD of INTS11 has a much more modest 
effect here than in the U7 assay and the authors say themselves that they think that Integrator is 
doing something besides just cleaving the RNA. 

Figure 7C. This is a very nice summary diagram but I have the suspicion that INTS10/13/14 plays a 
rather bigger role in some other function(s) in the cell. 
Typos/references 

Abstract. Line 5: form not from 

Page 18, line 4. Is 25 the right ref here? 

Throughout. Lots of stray commas and the English needs a bit of editing.