A gene needs to express itself in order to contribute to cellular functions. This requires information from the gene to be transcribed from DNA into an RNA molecule. Upon its transcription, each RNA molecule undergoes processing and passes through many stages of quality control and regulation, which are mediated by ribonucleoprotein complexes (RNPs).

Ribonucleoprotein complexes (RNPs) form when proteins bind to an RNA molecule, and they coordinate all of the regulatory stages that the RNA passes through. We develop techniques that integrate biochemistry and computational biology to obtain a comprehensive map of interactions between proteins and their RNA partners within our cells. We developed the individual-nucleotide resolution UV crosslinking and immunoprecipitation of protein-RNA complexes (iCLIP), and a related method called hiCLIP, which reveals the RNA-RNA contacts and higher-order conformation of RNPs. We use these methods to study how the sequence and structure of RNAs defines the composition and function of RNPs. A schematic of iCLIP and related protocols is shown below, and see also the recent comprehensive review of CLIP and complementary methods.

Cells can change their gene expression by modulating the composition of RNPs. Moreover, genetic studies have identified mutations that disrupt RNPs, which often cause neurologic diseases, particularly the motor neuron disease, also referred to as amyotrophic lateral sclerosis (ALS). We wish to understand how these mutations affect the assembly of RNPs, thereby initiating the molecular cascade leading to such diseases. In particular, we focus on protein-RNA condensates, which form through large numbers of weak links that are commonly affected by disease-causing mutations.

We study the following questions:

How do RNA-RNA, protein-RNA and protein-protein interactions guide the assembly of RNPs, and thereby coordinate RNA regulation?
How does RNA regulation contribute to brain development and function?
How do changes in RNA binding sites, especially those driven by transposable elements and cryptic splice sites, contribute to the evolution of RNA-protein networks?
How do various RNA features yield the multivalent interactions that enable the condensation of RNPs?
How do mutations or post-translational modifications in disordered regions of RNA-binding proteins alter their condensation propensity and thereby their RNA regulatory repertoire?
How does RNP condensation enable feedback regulation that promotes cellular homeostasis, and how is this perturbed upon aging and neurodegeneration?
How can we develop new therapeutic approaches to reestablish cellular homeostasis by modulating RNPs?

Main contributions to Science

Development of methods for RNA biology

To understand how RNPs regulate the life cycle of mRNAs in neurons, and how this can go wrong in diseases, it is crucial obtain detailed maps of protein-RNA binding sites in cells. We have developed several transcriptomic techniques for this purpose, including the nucleotide-resolution UV crosslinking and immunoprecipitation (iCLIP), which identifies protein-RNA contacts. To understand the role of RNA structure in RNP assembly, we also developed hybrid iCLIP (hiCLIP) that identifies RNA-RNA hybrids bound by double-strand RNA-binding proteins (dsRBPs), and informs on their roles in RNP assembly and function. We also develop computational tools to interpret the diverse types of high-throughput sequencing data to gain a comprehensive view of RNP assembly and functions within cells.

Iosub IA, Wilkins OG, Ule J. (2024) Riboseq-flow: A streamlined, reliable pipeline for ribosome profiling data analysis and quality control. Wellcome Open Res. Apr 11;9:179
West C, Capitanchik C, Cheshire C, Luscombe NM, Chakrabarti A, Ule J. (2023) nf-core/clipseq - a robust Nextflow pipeline for comprehensive CLIP data analysis. Wellcome Open Res. Jul 4;8:286.
Kuret K, Amalietti AG, Jones DM, Capitanchik C, Ule J. (2022) Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP. Genome Biol. Sep 9;23(1):191.
Michael Briese, Nejc Haberman, Chris Sibley,…,Jernej Ule (2019) A systems view of spliceosomal assembly and branchpoints with iCLIP, Nat Struct Mol Biol. Oct;26(10):930-940.
Haberman N*, Huppertz I*, Attig J, König J, Wang Z, Hauer C, Hentze MW, Kulozik AE, Le Hir H, Curk T, Sibley CR, Zarnack K*, Ule J* (2017) Insights into the design and interpretation of iCLIP experiments, Genome biology, Jan 16;18(1):7.
Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D’Ambrogio A, Luscombe N*, Ule J* (2015) hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature, Mar 26;519(7544):491-4.
Cereda M, Pozzoli U, Rot G, Juvan P, Schweitzer A, Clark T, Ule J. (2014) RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing. Genome Biol. Jan 31;15(1):R20.
Konig J, Zarnack K, Curk T, Gregor R, Kayikci M, Zupan,B, Luscombe NM, Ule J (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution, Nat. Struct. Mol. Biol., Jul;17(7):909-15

Mechanisms and functions of RNA processing

We have studied the positional principles whereby RNPs regulate RNA processing, which can be visualised as RNA maps of splicing and 3’ end processing regulation. This yielded insights into the importance of multivalent sequences, mechanisms of exon definition and recursive splicing, which informed on the mechanism and evolution of protein-RNA regulatory networks.

Ule J, Blencowe BJ. (2019) Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution. Mol Cell. Oct 17;76(2):329-345.
Modic M, Grosch M, Rot M, … Hirose T, Ule* J, Drukker* M (2018) Cross-regulation between TDP-43 and paraspeckles promotes pluripotency-differentiation transition, Mol Cell, Jun 6;74(5):951-965.e13
Blazquez L*, Emmett W, Faraway R, Pineda JMB, Bajew S, Gohr A, Haberman N, Sibley CR, Bradley RK, Irimia M, Ule J* (2018) Exon junction complex blocks recursive splicing to promote inclusion of RS-exons, Mol Cell, Nov 1;72(3):496-509.e9
Rot G*, Wang Z, Huppertz I, Modic M, Lenče T, Hallegger M, Haberman N, Curk T, von Mering C, Ule J*. (2017) High-Resolution RNA Maps Suggest Common Principles of Splicing and Polyadenylation Regulation by TDP-43. Cell Reports. May 2;19(5):1056-1067.
Sibley RC, Emmett W, Blazquez L, Faro A, Haberman N, Briese M, Trabzuni D, Ryten M, Hardy J, UK Brain Expression Consortium, Modic M, Curk T, Wilson SW, Plagnol V*, Ule J* (2015) Recursive splicing in long vertebrate genes. Nature, May 21;521(7552):371-5.

Regulation of transposon-derived RNA elements and its evolutionary implications

One of the surprises of our iCLIP studies was the major role that transposon-derived elements (TEs) play as hubs for RNP assembly. We have found that RNPs assemble on hundreds of thousands of TEs that are transcribed as part of host genes and thereby regulate RNA processing. Moreover, evolutionary changes TEs affect RNP assembly to drive the emergence of new tissue-specific exons, but mutations can also disrupt RNP assembly to cause diseases. Additionally, we uncovered recursive splice sites in the longest introns of human genes that are expressed in the brain, thus indicating a role for non-canonical splicing events in human transcripts. This opens the door to understand how variation in these elements across species, individuals and somatic tissues leads to changes in RNPs assembly and RNA regulation to facilitate evolutionary exploration of new gene functions.

Attig J*, Agostini F, Gooding C, Chakrabarti AM, Singh A, Haberman N, Zagalak JA, Emmett W, Smith CWJ, Luscombe NM, Ule J* (2018) Heteromeric RNP assembly at LINEs controls lineage-specific RNA processing, Cell, Aug 23;174(5):1067-1081.e17
Attig J, Ruiz de Los Mozos I, Haberman N, Wang Z, Emmett W, Zarnack K, König J*, Ule J* (2016) Splicing repression allows the gradual emergence of new Alu-exons in primate evolution. Elife. Nov 18;5
Zarnack K, König J, Tajnik M, Martincorena I, Stévant I, Reyes A, Anders S, Luscombe NM*, Ule J* (2013) Direct competition between hnRNP C and U2AF65 protects the transcriptome from the uncontrolled exonization of Alu elements. Cell, Jan 31;152(3):453-66

Protein-RNA complexes in neurodegeneration

We wish to understand how RNPs contribute to the mechanisms of neurodegeneration, and in particular Amyotrophic lateral sclerosis (ALS), a disease affecting motor neurons that is often caused by mutations that directly perturb the function of RNPs. We unravelled the transcriptome-wide functions of three RBPs, TDP-43, MATR3 and FUS, which are target of mutations causing ALS. We showed how an RNP called ‘paraspeckles’, which contains these three RBPs, plays a role in cell fate transitions through cross-regulation between TDP-43 and the scaffolding RNA called NEAT1. We also studied the roles of MATR3 and TDP-43 in repressing cryptic exons, such as those derived from transposable, which is important for RNA processing in the brain, with implications for evolution and disease.

We also showed how RNP condensation contributes to selective RNA binding and regulation, we studied mutations in the intrinsically disordered regions (IDR) of TDP-43 with a gradient of condensation propensities. We found that TDP-43 forms ‘binding-region condensates’ on RNAs, which requires unusually long clusters of RNA motifs. Changes in TDP-43 condensation modulate the regulation of a subset of transcripts that form these ‘binding-region condensates’. Thus, a network of RNAs can be selectively remodelled by changes in the properties of TDP-43’s condensates. This laid the mechanistic understanding that will allow to better understand how treatments could ameliorate the effects of these mutations.

Hallegger M*, Chakrabarti AM, Lee FCY, Lee BL, Amalietti AG, Odeh HM, Copley KE, Rubien JD, Portz B, Kuret K, Huppertz I, Rau F, Patani R, Fawzi NL, Shorter J, Luscombe NM, Ule J.* (2021) TDP-43 condensation properties specify its RNA-binding and regulatory repertoire, Cell. 2021 Sep 2;184(18):4680-4696.e22.
Soreq L; UK Brain Expression Consortium; North American Brain Expression Consortium, Rose J, Soreq E, Hardy J, Trabzuni D, Cookson MR, Smith C, Ryten M, Patani R*, Ule J* (2017) Major shifts in glial regional identity are a transcriptional hallmark of human brain aging, Cell Reports, Jan 10;18(2):557-570
Tollervey JR, Wang Z., Hortobágyi T, Witten J., Zarnack K., Kayikci M, Clark TA, Schweitzer AC, Rot G, Curk T, Zupan B, Rogelj B, Shaw CE, Ule J. (2011) Analysis of alternative splicing associated with aging and neurodegeneration in the human brain. Genome Research, Oct;21(10):1572-82.
Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. (2011) Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci;14(4):452-8.

Supramolecular RNP assembly and functions

Our insights from the RNA binding of TDP-43 demonstrated that its condensation promotes binding to highly multivalent RNA regions, which promotes homeostatic control of TDP-43 dosage via binding to its own mRNA. More recently, we reported on ‘interstasis’, a homeostatic mechanism in which increased concentration of diverse proteins within a RNA-protein condensate induces the sequestration of their own mRNAs. The selectivity of interstatic mRNA capture relies on the structure of the genetic code and conserved codon biases, which ensure that similar multivalent RNA regions encode similar low-complexity domains. Each type of multivalent RNA regions is bound by specific RNA-binding proteins, which form cooperative RNA-protein complexes through repetitive RNA interactions. We demonstrate the roles of TRA2 and their CLK-mediated phosphorylation in controlling the selective mRNA capture in nuclear speckles. Thus, we show that the condensation properties of nuclear speckles act as a sensor for interstasis, a collective negative-feedback loop that co-regulates mRNAs of highly dosage-sensitive genes, which primarily encode nuclear condensation-prone proteins.

We also study how Staufen proteins assemble on complex RNA structures that form through long-range duplexes in 3’UTRs, how dynamic such complex 3’UTR conformations are, and how they depend on the assembly of other RBPs.

Faraway R, Costello Heaven N, … Ule J (2025) Collective homeostasis of condensation-prone proteins via their mRNAs, Nature, in press
Hallegger M*, Chakrabarti AM, Lee FCY, Lee BL, Amalietti AG, Odeh HM, Copley KE, Rubien JD, Portz B, Kuret K, Huppertz I, Rau F, Patani R, Fawzi NL, Shorter J, Luscombe NM, Ule J.* (2021) TDP-43 condensation properties specify its RNA-binding and regulatory repertoire, Cell. 2021 Sep 2;184(18):4680-4696.e22.
Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D’Ambrogio A, Luscombe N*, Ule J* (2015) hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature, Mar 26;519(7544):491-4.

Physiologic signals and modifications that remodel RNPs

We are interested in the way that RNA regulation implements cellular signals, especially via phosphorylation and arginine methylation of RBPs. We showed how rapid changes in mRNA stability ensure clearance of the embryo's early programme of "naïve pluripotency". When the ERK signal is activated during embryo implantation, it triggers phosphorylation of LIN28A, which then binds to the terminal regions of mRNAs. There, LIN28A overlaps with binding of PABP, which binds to the same terminal regions already at early developmental stages. Once LIN28A docks to the sites bound by PABP, it rapidly triggers selective mRNA clearance.

Recently, we also showed that arginine methylation drives global 3′ UTR shortening, a hallmark of proliferating cells and tumour progression. This is reversed by inhibition of asymmetric dimethylation, and even more effectively by dual inhibition of symmetric and asymmetric dimethylation (DMAi), thus blocking production of oncogenic shortened isoforms. DMAi blocks 3′ UTR shortening in activated T cells, cancer cell lines and patient-derived organoids, and counteracts the impact of reduced CFIM25 expression. DMAi-induced APA results partly from impaired cleavage and polyadenylation activity due to decreased PCF11 levels, and regulated mRNAs contain characteristic signatures such as high GC-content, long separation between the affected poly(A) sites, and a position-dependent sequence code. These findings establish physiologic roles of PRMTs in promoting 3' UTR shortening.

Griffith L, Capitanchik C,… Ule J (2025) PRMT activity promotes global 3’ UTR shortening in proliferating cells, bioRxiv
Modic M, Kuret K, Steinhauser S, Faraway R, van Genderen E, Ruiz de Los Mozos I, Novljan J, Vičič Ž, Lee FCY, Ten Berge D, Luscombe NM, Ule J. (2024) Poised PABP-RNA hubs implement signal-dependent mRNA decay in development, Nature Str Mol Biol, Jul 25

We study the following questions:​

We study the following questions: