Intron Retention Controls Localization of lncRNAs PURPL and MALAT1 to Promote Cell Proliferation and Migration
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eLife Assessment
This manuscript provides important insights into how U2AF2-dependent intron retention regulates the localization and function of long noncoding RNAs, with convincing evidence supported by multiple complementary approaches. The work is notable for linking intron retention to nuclear speckle localization and cellular phenotypes, including proliferation and migration, although the mechanistic basis remains incompletely resolved. Overall, the study presents a compelling dataset with clear biological implications but would benefit from additional analyses to strengthen mechanistic interpretation and generality.
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Abstract
Intron retention (IR) is increasingly recognized as a feature of long noncoding RNAs (lncRNAs), yet the mechanisms that shape IR in lncRNAs and the functional consequences of this process remain largely unexplored. To investigate how IR contributes to lncRNA regulation, we performed a genome-wide screen to identify factors controlling IR in the lncRNA PURPL . This approach uncovered a prominent role for U2AF2, which promotes retention of a specific intron in PURPL through a weak polypyrimidine tract. IR of this intron drives nuclear enrichment of PURPL and enhances cell proliferation, revealing biological relevance. Transcriptome-wide analyses showed that although U2AF2 broadly supports canonical splicing consistent with its well-established function in promoting splicing, it also facilitates IR within a distinct subset of RNAs, including the nuclear speckle–associated lncRNA MALAT1 . Loss of U2AF2 disrupts MALAT1 speckle localization and using MALAT1 knockout cells reconstituted with wild-type or intron deleted variants, we identified a single intron critical for MALAT1 ’s speckle localization. Deletion of this intron from endogenous MALAT1 impaired speckle localization and reduced cell migration, phenocopying the loss of MALAT1 . Together, these findings reveal IR as a key regulatory mechanism governing lncRNA localization and function and uncover an unexpected role for U2AF2 in promoting IR within specific lncRNA contexts.
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eLife Assessment
This manuscript provides important insights into how U2AF2-dependent intron retention regulates the localization and function of long noncoding RNAs, with convincing evidence supported by multiple complementary approaches. The work is notable for linking intron retention to nuclear speckle localization and cellular phenotypes, including proliferation and migration, although the mechanistic basis remains incompletely resolved. Overall, the study presents a compelling dataset with clear biological implications but would benefit from additional analyses to strengthen mechanistic interpretation and generality.
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Reviewer #1 (Public review):
Intron retention is observed in many long noncoding RNAs. The authors here used a powerful genome-wide screening strategy to identify proteins controlling intron retention in the long noncoding RNA PURPL. One of the top hits across multiple cell lines surprisingly, was U2AF2, which is well known to bind the polypyrimidine tract close to the 3' splice site to promote splicing. Nonetheless, U2AF2 is working in the opposite direction here. Convincing follow-up RT-PCR experiments confirmed that knocking down U2AF2 does indeed lead to reduced intron retention of PURPL. The authors then show that this intron retention event is functionally important for both the nuclear retention of PURPL as well as its ability to enhance cell proliferation.
The authors then used transcriptome-wide analyses to look for additional …
Reviewer #1 (Public review):
Intron retention is observed in many long noncoding RNAs. The authors here used a powerful genome-wide screening strategy to identify proteins controlling intron retention in the long noncoding RNA PURPL. One of the top hits across multiple cell lines surprisingly, was U2AF2, which is well known to bind the polypyrimidine tract close to the 3' splice site to promote splicing. Nonetheless, U2AF2 is working in the opposite direction here. Convincing follow-up RT-PCR experiments confirmed that knocking down U2AF2 does indeed lead to reduced intron retention of PURPL. The authors then show that this intron retention event is functionally important for both the nuclear retention of PURPL as well as its ability to enhance cell proliferation.
The authors then used transcriptome-wide analyses to look for additional intron retention events affected by U2AF2. Among the ~250 genes with decreased intron retention (more splicing) upon U2AF2 knockdown was MALAT1, a well-established long noncoding RNA that normally localizes to nuclear speckles. Depletion of U2AF2 or removal of the MALAT1 2nd intron resulted in reduced speckle localization and cell migration, revealing a critical and fascinating role for this intron retention event. Overall, the authors have used a set of complementary approaches to clearly demonstrate a very intriguing role for U2AF2 in controlling intron retention and functionality of a set of long noncoding RNAs.
I feel the current work has revealed an important role of intron retention in controlling the localization and functionality of long noncoding RNAs, which is likely broad in scope and is likely regulated by cell state.
One experimental suggestion: The authors show that expressing intron-2 containing PURPL in PURPL-depleted cells is sufficient to induce faster proliferation, but a valuable comparison would be identifying the phenotype expressing spliced PURPL transcript.
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Reviewer #2 (Public review):
Summary:
This study identified U2AF1/2 as a regulator of pre-mRNA splicing that either promotes or supresses the splicing of introns on different genes. The authors then focused on two genes PURPL and MALAT1 that U2AF1/2 can promote intron retention of specific introns, and characterized the biological implications of these introns regulated by U2AF1/2.
Strengths:
(1) The experiments in this manuscript are relatively rigorously designed and performed, often with validation checks such as verifying the knockout, verifying the treatment itself doesn't have an effect, etc.
(2) The experiments provided comprehensive support for the claims that these specific introns are important for the stability or nuclear localization of the RNA, as well as that U2AF1/2 suppresses the splicing of these introns.
(3) The …
Reviewer #2 (Public review):
Summary:
This study identified U2AF1/2 as a regulator of pre-mRNA splicing that either promotes or supresses the splicing of introns on different genes. The authors then focused on two genes PURPL and MALAT1 that U2AF1/2 can promote intron retention of specific introns, and characterized the biological implications of these introns regulated by U2AF1/2.
Strengths:
(1) The experiments in this manuscript are relatively rigorously designed and performed, often with validation checks such as verifying the knockout, verifying the treatment itself doesn't have an effect, etc.
(2) The experiments provided comprehensive support for the claims that these specific introns are important for the stability or nuclear localization of the RNA, as well as that U2AF1/2 suppresses the splicing of these introns.
(3) The writing of the manuscript is very clear and doesn't overstate the conclusions that can be drawn from the experiments.
Weaknesses:
I think one main weakness of this study is the lack of a deeper analysis of the mechanisms. Whether studying the mechanism is within the scope of this paper is probably debatable, but with the current experiment setup and data, I believe there are some analyses that can be relatively easily done to enhance the value or significance of this study. My detailed questions and suggestions are listed below:
(1) Line 194-195 and Figure 2A: How many RBPs are included in "other RBPs" in line 194? Does "other RBPs" only include PTBP1, PRPF8 and SRSF1 in Figure 2A, or do they include all the ~100 RBPs with HepG2 eCLIP data available on ENCODE? If U2AF1/2 have the highest occupancy around the intron 2 region among the ~100 RBPs, it would be nice to visualize it.
(2) Figure 2A and 2B: Why didn't U2AF2 show interaction with exon 2 and 3 in RNA-IP but showed enrichment over exon 2 and exon 3 regions in the eCLIP data?
(3) Figure 3C - 3F: Maybe I misinterpreted the experiments, but to my understanding, these experiments showed that the exogenous PURPL with intron 2 promoted cell proliferation compared to when the exogenous PURPL wasn't induced, but didn't compare to the effect of the same amount of PURPL with intron 2 removed. Wouldn't it be clearer to compare the effects of exogenous PURPL with intron 2 and exogenous PURPL without intron 2 to pinpoint whether the effect is related to intron 2? Without an intron 2 specific experiment, these current experiments don't seem to provide much added value than "PURPL promotes cell proliferation".
(4) It's not very clear what proportion of these introns are retained in the endogenous PURPL and MALAT1 in various tissues, cell types and conditions. I think it will be valuable to provide this background (either from previous research, public database or data from this study).
(5) Since U2AF1/2 have a wide range of targets as demonstrated by Figure 4A, I think it would be valuable to have some experiments that directly disrupt the interaction between U2AF1/2 and PURPL and MALAT1 and test the effect on splicing outcomes, such as by mutating the sequence that U2AF1/2 bind to. The section on the weak py-tract of PURPL touched upon this topic but focused more on how the weak py-tract causes the intron 2 retention in the background rather than how U2AF1/2 binding and action were affected by sequence mutations. I think experiments on disrupting the direct binding between U2AF1/2 on targets can provide valuable mechanistic insights.
(6) Across all the target genes of U2AF1/2, it might be feasible to do some systematic analysis to find what correlates with whether U2AF1/2 have a promoting or suppressing effect on intron splicing. For example, do genes with decreased IR after U2AF2 depletion systematically have a weak py-tract compared to genes with increased IR? This dataset can potentially provide many hypotheses for understanding the dual role of U2AF1/2.
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Reviewer #3 (Public review):
Summary:
This manuscript characterized the splicing regulation of two long non-coding RNAs relevant to cancer, starting with a focus on PURPL and ending with insights into MALAT1. A CRISPR screen for the regulators of PURPL intron retention revealed a role for the U2AF heterodimer in inducing this retention, with U2AF2 as the actual hit. This is surprising, because the canonical function of U2AF is to recognize the polypyrimidine tract (PPT) and 3' splice site junction to induce splicing at the site. The brief mechanistic characterization of this phenomenon showed that this intron retention accounts for the nuclear localization and instability of the PURPL transcript, and seems to confer the enhanced cell proliferation feature. U2AF2 also induces retention of two introns in MALAT1, and one of them is …
Reviewer #3 (Public review):
Summary:
This manuscript characterized the splicing regulation of two long non-coding RNAs relevant to cancer, starting with a focus on PURPL and ending with insights into MALAT1. A CRISPR screen for the regulators of PURPL intron retention revealed a role for the U2AF heterodimer in inducing this retention, with U2AF2 as the actual hit. This is surprising, because the canonical function of U2AF is to recognize the polypyrimidine tract (PPT) and 3' splice site junction to induce splicing at the site. The brief mechanistic characterization of this phenomenon showed that this intron retention accounts for the nuclear localization and instability of the PURPL transcript, and seems to confer the enhanced cell proliferation feature. U2AF2 also induces retention of two introns in MALAT1, and one of them is essential for its nuclear speckle localization and enhanced cell migration.
Strengths:
These findings about PURPL and MALAT1 are clear and interesting.
Weaknesses:
The results are not sufficiently connected to each other, because one regulation is nuclear-speckle dependent but not the other.
Here are my specific comments:
Major comments:
The main issue is the lack of focus because of the distinct and incomplete analysis pertaining to the two long noncoding RNAs, PURPL and MALAT1. The paper starts with a very good genetic screen on the former, and immunofluorescence and functional analysis on the latter, with U2AF2 as the main link to induce intron retention. The first one does not show clear localization while the second docks to nuclear speckles, apparently because of the retained intron. Hence the two mechanisms are related yet distinct. Here are some suggestions to enhance the characterization and connection between the two cases:
(1) As the MALAT1 intron 2 retention contributes to its speckle localization but not the retained PURPL intron, the retained introns or their 3' splice site sequences should be swapped to see if they determine the localization.
(2) Figure 3, the rescue of the PURPL knockout by the intron-retained RNA to induce proliferation is a powerful experiment, that is lacking the rescue with the RNA without the intron as a control. This must be done and shown.
(3) The weakness of the PPT of PURPL intron 2 appears as a clear feature of its retention dependent on U2AF2, which appears direct, as backed by CLIP data. It would be good to show direct binding by EMSA or equivalent techniques. Furthermore, the data is also consistent with other determinants. The exon and upstream intronic sequences, including the branch point, could also be involved, so mutations in these are also required.
(4) In brief, what are the commonalities and differences between PURPL and MALAT1 with regard to their U2AF2-dependent intron retention?
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