sCLIP-an integrated platform to study RNA-protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs.

RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here, we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally, it permits a radiolabel-free visualization of immunoprecipitated RNA. In a proof of concept, we identify that CSTF2tau binds many previously not recognized RNAs including histone, snoRNA and snRNAs. CSTF2tau-binding is associated with internal oligoadenylation resulting in shortened snRNA isoforms subjected to rapid degradation. We provide evidence for a new mechanism whereby CSTF2tau controls the abundance of snRNAs resulting in alternative splicing of several RNAs including ANK2 with critical roles in tumorigenesis and cardiac function. Combined with a bioinformatic pipeline sCLIP thus uncovers new functions for established RBPs and fosters the illumination of RBP-protein interaction landscapes in health and disease.

Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.

Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3(-/-) mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation-induced immune suppression. These findings establish that UVB damage is detected by TLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.

Trimethylguanosine nucleoside inhibits cross-linking between Snurportin 1 and m3G-CAPPED U1 snRNA.

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin alpha/beta. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import.

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

OBJECTIVE: To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. METHODS: To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. RESULTS: Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. CONCLUSION: These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

A UV-crosslinkable interaction in human U6 snRNA.

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25-99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5' deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3' of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.

Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein.

Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size. The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end. They are labeled in the presence of [alpha-32P]UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts. That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously. Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches. This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein. Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli. In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP.

Association of U6 snRNA with the 5'-splice site region of pre-mRNA in the spliceosome.

U6 snRNA is one of the five RNA species required for splicing of nuclear pre-mRNAs. High conservation of its sequence has led to the hypothesis that U6 snRNA plays a catalytic role in splicing. If this is the case, U6 snRNA should be localized close to sites where the splicing reaction occurs. However, this has never been demonstrated. Here, we have shown that U6 snRNA is cross-linked to the 5'-splice site region of pre-mRNA by UV irradiation during the in vitro splicing reaction. We have also detected the cross-link of U6 snRNA and the region around the branchpoint of the intron lariat. The results show that U6 snRNA is present near the splice sites in the splicing reaction and support the idea that U6 snRNA is a catalytic element in the spliceosome.

In vivo UV cross-linking of U snRNAs that participate in trypanosome trans-splicing.

The maturation of mRNAs in Trypanosoma brucei involves a trans-splicing reaction whereby the 5' 39 nucleotides of a small RNA, called the spliced leader (SL) RNA, are joined with a pre-mRNA transcript. The trans-splicing reaction appears mechanistically similar to cis-splicing of nuclear pre-mRNAs, and homologs of the U2, U4, and U6 snRNAs are required for the process. In the work presented here, potential RNA-RNA interactions between the SL RNA and the U snRNAs of trypanosomes were examined by UV light induction of RNA-RNA cross-links in vivo. We detected cross-linkage between U2 and U6 RNAs and, as might be expected, between the trypanosome U4 and U6 RNAs. The latter contain extensive sequence complementarity and are thought to exist predominantly in a single RNP. We also detected an SL RNA species following in vivo UV treatment, which may represent either an intramolecular cross-link in the SL RNA or a cross-link formed between the SL RNA and an as yet unidentified small RNA. Mapping of the cross-link position between U2 and U6 RNAs is consistent with base-pairing between the 5' domain of U2 and the 3' end of U6 RNA. These results reveal the existence, in vivo, of cognate RNA-RNA interactions in the RNA homologs that participate in trans-splicing in trypanosomes and cis-splicing in other eukaryotes.

RNA synthesis and stability in UV-irradiated and nonirradiated mouse L cells.

In mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleu was also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Direct cross-linking of snRNP proteins F and 70K to snRNAs by ultra-violet radiation in situ.

Protein-RNA interactions in small nuclear ribonucleoproteins (UsnRNPs) from HeLa cells were investigated by irradiation of purified nucleoplasmic snRNPs U1 to U6 with UV light at 254 nm. The cross-linked proteins were analyzed on one- and two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage was demonstrated by isolating protein-oligonucleotide complexes from snRNPs containing 32P-labelled snRNAs after exhaustive digestion with a mixture of RNases of different specificities. The primary target of the UV-light induced cross-linking reaction between protein and RNA was protein F. It was also found to be cross-linked to U1 snRNA in purified U1 snRNPs. Protein F is known to be one of the common snRNP proteins, which together with D, E and G protect a 15-25 nucleotide long stretch of snRNAs U1, U2, U4 and U5, the so-called domain A or Sm binding site against nuclease digestion (Liautard et al., 1982). It is therefore likely that the core-protein may bind directly and specifically to the common snRNA domain A, or else to a sub-region of this. The second protein which was demonstrated to be cross-linked to snRNA was the U1 specific protein 70K. Since it has been shown that binding of protein 70K to U1 RNP requires the presence of the 5' stem and loop of U1 RNA (Hamm et al., 1987) it is likely that the 70K protein directly interacts with a sub-region of the first stem loop structure.

Metabolism of U6 RNA species in nonirradiated and UV-irradiated mammalian cells.

We observed a series of rapidly labeled U6 RNA bands, which were hybrid selected with U6 DNA, in nonirradiated human cells. The electrophoretic mobility of these bands in denaturing gels was lower than that of the known mature U6 RNA species, and was equivalent to transcripts up to approximately 7 nucleotides longer. These multiple U6 RNA species lost their label during a chase without a proportional increase in radioactivity in the known mature U6 RNA, which suggests that a substantial fraction is not processed into the major mature U6 RNA. During a label chase, the multiple U6 RNA bands appeared first in the cytoplasmic fraction and later in nuclei. One of the major rapidly labeled U6 RNA bands had the electrophoretic mobility of an RNA species one nucleotide shorter than the known mature U6 RNA. UV light induced a UV dose-dependent, preferential disappearance of recently synthesized molecules of the U6 RNA species of higher gel electrophoretic mobility, including the known mature U6 RNA. Since this effect was seen in cells pulse-labeled immediately before or after irradiation, it suggests that UV radiation induces the specific degradation of the electrophoretically faster moving species of U6 RNA, which are apparently shorter chains. The effect of UV light was RNA species-specific, was not seen in molecules synthesized long (e.g., 22 hr) before irradiation, and occurred in human and mouse cells.

Effect of ultraviolet light on the expression of genes for human U1 RNA.

Two types of UV-light-induced inhibitions of the synthesis of small nuclear RNA species U1, U2, U3, U4, and U5 were described previously: an immediate inhibition and a separate, delayed suppression that requires 1-2 hr of postirradiation cell incubation and UV doses that are about tenfold lower. In the present report, U1 RNA transcription in isolated nuclei from HeLa cells, assayed by RNAase T1 protection, reproduced the delayed inhibition. The sizes of the protected RNA fragments suggest that it is the initiation of U1 RNA transcription that is blocked during this inhibition. Transient expression of a marked human U1 RNA gene that contains 425 and 92 nucleotides of the 5' and 3' flanking sequences, respectively, showed delayed, but not immediate inhibition (while the endogenous U1 RNA genes exhibited immediate suppression). This indicates that continuity of the U1 gene flanking sequences beyond those segments and/or chromosomal integration of the U1 gene are not needed for the delayed inhibition, but may be required for the immediate inhibition. Irradiation of a U1 RNA gene, followed by its injection into Xenopus laevis oocyte nuclei, did not reproduce the immediate or delayed inhibitions. This suggests that direct UV radiation damage to DNA in the U1 RNA gene region is not the critical lesion in either the immediate or delayed UV-light-induced inhibitions of U1 RNA synthesis. In addition, the RNAase T1 protection pattern of transcripts synthesized in isolated nuclei from nonirradiated HeLa cells suggests that these cells may produce small amounts of U1 RNA molecules with variant nucleotide sequences in the mature region of the transcript.

Inhibition of small nuclear RNA synthesis by ultraviolet radiation.

It was reported earlier that the biosynthesis of small nuclear RNAs (snRNAs) (U1, U2, U3, U4, and U5) shows an unexpected great sensitivity to ultraviolet (UV) radiation (254 nm). In this "early" inhibition, snRNA formation is suppressed immediately after exposure to UV light. There is also a second "late" inhibition of snRNA biosynthesis which requires lower doses of UV radiation and 1-2 h of postirradiation cell incubation to develop fully. In the present work we asked which step, within the metabolic pathway leading to the accumulation of newly made snRNA, is affected by UV light. Both for the early and late UV radiation-induced inhibitions: (a) similar results were obtained after pulse labeling or pulse chasing the radiolabel, implying that UV light did not decrease the stability of newly made snRNA; and (b) gel electrophoretic analysis of radiolabeled RNA that had been hybrid selected with cloned snRNA genes showed no accumulation of putative snRNA precursors, suggesting that UV radiation did not block snRNA processing. Instead, when transcription was carried out in isolated nuclei from irradiated cells, the effects of "early" and "late" inhibition were reproduced, indicating that transcription was affected. The early suppression appears to be a separate reaction from the late inhibition, since U1 snRNA transcription in isolated nuclei was inhibited in the absence of postirradiation cell incubation. There is a small fraction of snRNA synthesis that is resistant to high UV light doses (greater than or equal to 870 J/m2) right after irradiation, but is sensitive to lower doses (less than or equal to 36 J/m2) when the cells are incubated for 2 h after irradiation.

Effect of UV light on small nuclear RNA synthesis: increased inhibition during postirradiation cell incubation.

It has been shown previously that the synthesis of small nuclear RNAs (snRNAs) U1, U2, U3, U4, and U5, in contrast to that of all other RNA species tested, decreases markedly within 2 h of cell incubation after exposure to UV light (254 nm), while pyrimidine dimers are being removed from DNA. We examined the possibility that the postirradiation cell incubation-dependent, UV light-induced inhibition of snRNA synthesis might reflect hypersensitivity of the snRNA transcriptional domains to single-stranded DNA nicks or relaxation of DNA torsional stress or both that occur during DNA repair. This late suppression of snRNA biosynthesis was as pronounced in UV light-irradiated (DNA incision-deficient) xeroderma pigmentosum fibroblasts (complementation group A) as in irradiated normal human fibroblasts. The synthesis of snRNAs was not preferentially sensitive to gamma radiation (which produces single-stranded DNA breaks) or novobiocin or nalidixic acid (which induce DNA relaxation). Neither of these two drugs prevented the UV light-induced inhibition of snRNA synthesis observed during postirradiation cell incubation. These results suggest that the late suppression of snRNA synthesis does not result from hypersensitivity of snRNA transcriptional domains to single-stranded DNA cleavages or relaxation of DNA torsional strain. The UV light-induced late inhibition of snRNA synthesis: shows an inactivation curve whose slope differs from that observed immediately after irradiation; is seen in untransformed cells as well as established cells lines; and has been conserved between birds and mammals.