Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles
2012; Springer Nature; Volume: 31; Issue: 20 Linguagem: Inglês
10.1038/emboj.2012.251
ISSN1460-2075
AutoresTakao Naganuma, Shinichi Nakagawa, Akie Tanigawa, Y. Sasaki, Naoki Goshima, Tetsuro Hirose,
Tópico(s)RNA modifications and cancer
ResumoArticle7 September 2012free access Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles Takao Naganuma Takao Naganuma Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Shinichi Nakagawa Shinichi Nakagawa RNA Biology Laboratory, RIKEN Advanced Science Institute, Wako, Japan Search for more papers by this author Akie Tanigawa Akie Tanigawa Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Yasnory F Sasaki Yasnory F Sasaki Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Naoki Goshima Naoki Goshima Biological Systems Control Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Tetsuro Hirose Corresponding Author Tetsuro Hirose Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Takao Naganuma Takao Naganuma Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Shinichi Nakagawa Shinichi Nakagawa RNA Biology Laboratory, RIKEN Advanced Science Institute, Wako, Japan Search for more papers by this author Akie Tanigawa Akie Tanigawa Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Yasnory F Sasaki Yasnory F Sasaki Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Naoki Goshima Naoki Goshima Biological Systems Control Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Tetsuro Hirose Corresponding Author Tetsuro Hirose Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan Search for more papers by this author Author Information Takao Naganuma1, Shinichi Nakagawa2, Akie Tanigawa1, Yasnory F Sasaki1, Naoki Goshima3 and Tetsuro Hirose 1 1Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan 2RNA Biology Laboratory, RIKEN Advanced Science Institute, Wako, Japan 3Biological Systems Control Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan *Corresponding author. Functional RNomics Team, Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koutou, Tokyo 135-0064, Japan. Tel.: +81 3 3599 8521; Fax: +81 3 3599 8579; E-mail: [email protected] The EMBO Journal (2012)31:4020-4034https://doi.org/10.1038/emboj.2012.251 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3′-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3′-end polyadenylation of the NEAT1_1 isoform. An in vitro 3′-end processing assay revealed that HNRNPK arrested binding of the CPSF6–NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. Introduction Recent postgenomic transcriptome analyses reveal that many nonprotein-coding transcripts, so-called noncoding RNAs (ncRNAs), are transcribed from large portions of mammalian genomes (Carninci et al, 2005; Kapranov et al, 2007). The limited numbers of long ncRNAs that have been characterized exhibit diverse functions, as well as cell type-specific expression and localization to subcellular compartments (Prasanth and Spector, 2007; Mercer et al, 2009; Wang and Chang, 2011). Most of the newly discovered ncRNAs are likely transcribed by RNA polymerase II. However, extensive analyses of the subcellular localization of human transcripts reveal that ncRNAs are enriched in the cell nucleus, suggesting that they play diverse roles in nuclear events (Kapranov et al, 2007; Prasanth and Spector, 2007). The mammalian cell nucleus is highly organized. It is composed of distinct nuclear bodies that contain proteins or RNAs characteristic of particular nuclear processes. To date, ∼10 different nuclear bodies have been characterized (Spector, 2006). Several long ncRNAs, such as Xist, Gomafu (Miat), Malat1 (NEAT2), NEAT1 (MENε/β), TUG1, and GRC-RNAs, localize to specific nuclear bodies (Clemson et al, 1996, 2009; Hutchinson et al, 2007; Sone et al, 2007; Sasaki et al, 2009; Sunwoo et al, 2009; Zheng et al, 2010; Yang et al, 2011). In particular, Malat1 localizes to nuclear speckles, where it regulates alternative splicing by modulating the phosphorylation status of Serine/Arginine (SR)-splicing factors (Tripathi et al, 2010). Malat1 controls growth signal-responsive gene expression through its association with unmethylated polycomb 2 protein (Yang et al, 2011). Paraspeckles are recently discovered nuclear bodies that are usually detected in cultured cell lines as a variable number of foci found in close proximity to the nuclear speckles. Paraspeckles contain characteristic RNA-binding proteins, including paraspeckle protein 1 (PSPC1), RBM14, NONO, CPSF6, and SFPQ (Fox et al, 2002; Dettwiler et al, 2004; Prasanth et al, 2005). PSPC1, NONO, and SFPQ share common domain structures comprised of two RNA-recognition motifs (RRMs). Collectively, these three proteins comprise the Drosophila melanogaster behaviour and human splicing (DBHS) protein family (Bond and Fox, 2009). The discovery of the specific paraspeckle localization of NEAT1 ncRNA opened a new window in paraspeckle research (Chen and Carmichael, 2009; Clemson et al, 2009; Sasaki et al, 2009; Sunwoo et al, 2009). NEAT1 ncRNA are transcribed from a genetic locus called familial tumour syndrome multiple endocrine neoplasia (MEN) type I on human chromosome 11 (Guru et al, 1997) and are comprised of two isoform transcripts, 3.7-kb NEAT1_1 (MENε) and 23-kb NEAT1_2 (MENβ). Both RNAs are produced from the same promoter. Alternatively, they can be processed at the 3′-end to produce a canonically polyadenylated NEAT1_1 and a noncanonically processed NEAT1_2. RNase P recognizes the tRNA-like structure and cleaves it to form the nonpolyadenylated 3′-end of NEAT1_2 (Sunwoo et al, 2009). The knockdown of NEAT1 ncRNA leads to the disintegration of paraspeckles, suggesting that these ncRNAs serve as a core structural component (Chen and Carmichael, 2009; Clemson et al, 2009; Sasaki et al, 2009; Sunwoo et al, 2009). However, the biological function of paraspeckles and the role(s) of NEAT1 ncRNA remain to be elucidated. We recently found that paraspeckles were not essential for viability and development in a mouse model under normal conditions, suggesting that they play roles under certain stress conditions (Nakagawa et al, 2011). It has been noted that CTN-RNA, an isoform of mCat2 mRNA, is retained specifically in the paraspeckle. Intriguingly, the long 3′-untranslated region (UTR) of CTN-RNA is cleaved by an unidentified endoribonuclease upon exposure to certain stresses, which leads to the export of processed mCat2 mRNA for cytoplasmic translation (Prasanth et al, 2005). The CTN-RNA 3′-UTR contains a long inverted-repeat sequence that is capable of forming intramolecular double-stranded RNAs that are A-to-I edited. The hyperedited CTN-RNAs are enriched in the paraspeckles. Thus, paraspeckles are thought to suppress the expression of hyperedited transcripts through nuclear retention (Prasanth et al, 2005). Inverted Alu repeat sequences are commonly found in the 3′-UTRs of multiple mRNAs in human cells (Chen et al, 2008). This finding suggests that the expression of these transcripts is suppressed by a nuclear retention mechanism. We previously reported that two paraspeckle-localized DBHS family proteins, SFPQ and NONO, are required for paraspeckle integrity and for the accumulation of NEAT1_2 but not NEAT1_1 (Sasaki et al, 2009). These results suggest that NEAT1_1 alone is unable to maintain paraspeckle integrity. By contrast, overexpressed NEAT1_1 is reportedly capable of increasing the number of paraspeckles, which suggests that it is the functional isoform for paraspeckle formation (Clemson et al, 2009). An electron microscopic study revealed the location of the NEAT1_2 and NEAT1_1 isoforms. The common NEAT1 region and NEAT1_2 3′-terminal region were located at the paraspeckle periphery, whereas the NEAT1_2 middle region was located in the paraspeckle interior. These findings suggest the importance of NEAT1_2 for the maintenance of paraspeckle integrity (Souquere et al, 2010). In this study, the essential components for paraspeckle formation were determined. Plasmid rescue experiments revealed that NEAT1_2 but not NEAT1_1 is a necessary RNA for de novo paraspeckle formation. To analyse the detailed process of paraspeckle formation, we sought to identify unknown paraspeckle components. RNAi analyses identified additional factors, each with distinct roles, which were indispensible for paraspeckle formation. One of the essential PSPs was involved in the alternative 3′-end processing of NEAT1. This protein arrested the canonical NEAT1_1 3′-end processing, which led to preferential selection for the noncanonical processing of the NEAT1_2 3′-end. Our data provide important insights into the process of paraspeckle formation on the specific nuclear-retained long ncRNAs. Results The NEAT1_2 ncRNA isoform is essential for paraspeckle formation We first attempted to clarify which NEAT1 isoform(s) were required for de novo paraspeckle formation. MEFs were prepared from NEAT1 knockout mice (MEF−/−) (Nakagawa et al, 2011), in which paraspeckles were absent, for rescue experiments with the expression plasmid of either the NEAT1_1 or NEAT1_2 isoform. The expression levels of NEAT1_1 and NEAT1_2 from the plasmids were comparable (Supplementary Figure S1C). Many of the paraspeckle-like foci that were detectable with both anti-SFPQ antibody immunostaining and NEAT1 RNA-FISH appeared when NEAT1_2 but not NEAT1_1 was transiently expressed from the plasmid (Figure 1A; Supplementary Figure S1A and B). This result indicates that NEAT1_2 is an authentic RNA component that is capable of de novo paraspeckle formation. Figure 1.NEAT1_2 is a potent RNA component for paraspeckle formation. (A) NEAT1_2 but not NEAT1_1 rescues paraspeckle formation. Intact paraspeckles were detected by RNA-FISH with the antisense probe of mouse NEAT1 ncRNA and coimmunostaining of endogenous SFPQ. Paraspeckles, which were observed in WT MEF cells, were undetectable in MEF cells prepared from NEAT1-knockout mice (KO). Paraspeckles were detected in KO-MEF cells transfected with a plasmid expressing NEAT1_1 (KO+NEAT1_1) or NEAT1_2 (KO+NEAT1_2). (B) Effect of actinomycin D treatment on the reformed paraspeckle-like foci. KO-MEF cells were cotransfected with plasmids expressing NEAT1_2 ncRNA and SFPQ–Flag. Transfected cells were treated with 0.3 μg/ml actinomycin D for 4 h. Reformed paraspeckle-like foci were visualized with RNA-FISH of NEAT1 and coimmunostained with anti-Flag M2 antibody. (C) NEAT1_2 ncRNA is more competent than NEAT1_1 ncRNA at elevating the number of paraspeckles. NIH3T3 cells were cotransfected with expression plasmids of control (+control), NEAT1_1 (+NEAT1_1), or NEAT1_2 (+NEAT1_2), together with SFPQ–Flag. The counted paraspeckle numbers are shown in Supplementary Figure S1D. Scale bar, 10 μm. Download figure Download PowerPoint To prove that the rescued foci exhibited characteristics common to endogenous paraspeckles, transfected MEF(−/−) were treated with actinomycin D. Rescued foci did not appear with actinomycin D treatment. Instead, SFPQ–Flag (as a cotransfected marker) and endogenous PSPs relocated to perinucleolar caps, from which NEAT1_2 ncRNA was absent (Figure 1B). Paraspeckles reportedly display actinomycin D-induced disruption and the concomitant relocation of protein components (Fox et al, 2002; Shav-Tal et al, 2005). In the present study, the overexpression of either NEAT1_2 or NEAT1_1 in NIH3T3 cells led to elevated nuclear paraspeckle numbers; however, NEAT1_2 was more stimulatory than NEAT1_1 (Figure 1C; Supplementary Figure S1D). Taken together, these results indicate that the NEAT1_2 isoform is an essential RNA component for paraspeckle formation, and the NEAT1_1 isoform is not essential but can contribute to paraspeckle formation only when the NEAT1_2 isoform is present. Identification of new paraspeckle components We previously reported that two RNA-binding proteins that are essential for paraspeckle formation, NONO and SFPQ, preferentially bind to and stabilize the NEAT1_2 isoform (Sasaki et al, 2009). To obtain further insights into the paraspeckle structure, additional PSPs were searched for by employing the human full-length cDNA resource (FLJ Clones) available from the authors’ affiliate (Maruyama et al, 2012). In this cDNA collection, the intact protein-coding regions of 18 467 human proteins are fused with Venus fluorescent protein. FLJ Clones provides information concerning the intracellular localization of >18 000 human proteins, through the transfection of each cDNA clone (Figure 2A). Figure 2.Identification of novel PSP components. (A) Experimental strategy to identify new PSPs. (B) Selection of FLJ-Venus clones that localize to paraspeckle-like nuclear foci. Paraspeckles were visualized by the immunostaining of SFPQ. Three representatives (PSP10, PSP14, and PSP33) of the new PSPs are shown. (C) Confirmation of the paraspeckle localization of endogenous PSP counterparts. Antibodies against each counterpart of PSP10, PSP14, and PSP33 (EWSR1, FUS and TAF15, respectively) were employed to monitor the localization (see Supplementary Table S4). NEAT1 ncRNA was used as a paraspeckle marker. (D) Effect of actinomycin D treatment on the localization patterns of selected PSPs. Localization of the Venus clones in B was monitored after actinomycin D treatment. SFPQ is an endogenous PSP control. Data regarding other PSPs are shown in Supplementary Figures S2 and S3. Scale bar, 10 μm. Download figure Download PowerPoint Initially, 68 cDNA clones whose products exhibited the typical localization pattern of paraspeckle-like nuclear foci were selected. The identities of the foci were determined by immunostaining the endogenous SFPQ, to see if the Venus signals overlapped with the SFPQ signals (Figure 2B; Supplementary Figure S2A; Supplementary Table S1) but not with the signals of other nuclear bodies (Supplementary Figure S3B). This screening led to the eventual selection of 34 cDNA clones. Endogenous proteins corresponding to the identified cDNA clones were immunostained with their respective antibodies, when available (Figure 2C; Supplementary Figure S2B; Supplementary Table S1). The correct paraspeckle localization of all 27 examined proteins was confirmed, and no false positives were identified. As a second screen, we confirmed the relocation of each Venus-fusion protein upon actinomycin D treatment. All 34 fusion proteins relocated to the perinucleolar caps (Supplementary Table S1). These caps corresponded to the destination of the endogenous PSPs (Figure 2D; Supplementary Figure S3A), but were distinct from those of the nucleolar or Cajal body proteins (Supplementary Figure S3B). Therefore, 34 cDNA clones, designated PSP3 through PSP36, were confirmed as new PSPs. Additionally, TARDBP (TDP43), which was recently reported to interact prominently with NEAT1 ncRNA in the brain from frontotemporal lobar degeneration (FTLD) patients (Tollervey et al, 2011), was confirmed to localize to the paraspeckle in HeLa cells by immunostaining of endogenous protein (Supplementary Figure S2B) and detection of TARDBP-Venus localization (Supplementary Figure S2A). A comparison of all of the PSPs (Table I; Figure 4) indicated that most possessed canonical RNA-binding domains (Burd and Dreyfuss, 1994): 20 proteins with RRMs, two proteins with KH motifs, and five proteins with RGG boxes. Eight proteins possessed one or more zinc-finger motifs, which are involved in RNA binding (Brown, 2005). Three related RNA-binding proteins, FUS, EWSR1, and TAF15, which are known as significant disease-related proteins (Law et al, 2006), were all identified as PSPs. We also identified CPSF6, NUDT21, and CPSF7, which are components of the CFIm complex that regulates the 3′-end processing of mRNA, as PSPs. Table 1. Paraspeckle proteins Proteins Accession RNA-binding motifs Other motifs Category PSP # HUGO Synonyms New paraspeckle proteins PSP3 AHDC1 Q5TGY3 AT hook 3B PSP4 AKAP8L Q9ULX6 2 ZnF C2H2s 3B PSP5 CELF6 BRUNOL6 Q96J87 3 RRMs ND PSP6 CIRBP Q14011 RRM 3B PSP7 CPSF7 CFIm59 Q8N684 RRM 2 PSP8 DAZAP1 Q96EP5 2 RRMs 1B PSP9 DLX3 O60479 homeodomain ND PSP10 EWSR1 Q01844 RRM ZnF RanBP2 3B PSP11 FAM98A Q8NCA5 2 PSP12 FAM113A Q9H1Q7 2 PSP13 FIGN Q5HY92 2 PSP14 FUS TLS P35637 RRM ZnF RanBP2 1B PSP15 HNRNPA1 P09651 2 RRMs 2 PSP16 HNRNPA1L2 Q32P51 2 RRMs ND PSP17 HNRNPF P52597 3 RRMs ND PSP18 HNRNPH1 P31943 3 RRMs ND PSP19 HNRNPH3 P31942 2 RRMs 1B PSP20 HNRNPK P61978 3 KHs 1A PSP21 HNRNPR O43390 3 RRMs 2 PSP22 HNRNPUL1 Q9BUJ2 SAP, SPRY 2 PSP23 MEX3C Q5U5Q3 2 KHs ZnF RING ND PSP24 NUDT21 CFIm25 O43809 NUDIX hydrolase 3A PSP25 RBM3 P98179 RRM 3B PSP26 RBM4B RBM30 Q9BQ04 2 RRMs ZnF CCHC 3B PSP27 RBM7 Q9Y580 RRM 3B PSP28 RBM12 Q9NTZ6 3 RRMs 2 PSP29 RBMX P38159 RRM 3B PSP30 RUNX3 Q13761 3B PSP31 SRSF10 FUSIP1, SRp38 O75494 RRM RS 2 PSP32 SS18L1 O75177 ND PSP33 TAF15 Q92804 RRM ZnF RanBP2 3B PSP34 UBAP2L Q14157 3A PSP35 ZC3H6 P61129 3 ZnF C3H1s 3B PSP36 ZNF335 Q9H4Z2 13ZnF C2H2s 3B TARDBP TDP43, ALS10 Q13148 2 RRMs 2 Known paraspeckle proteins CPSF6 CFIm68 Q16630 RRM 3A NONO p54nrb Q15233 2 RRMs 1A PSPC1 PSP1 Q8WXF1 2 RRMs 3B RBM14 PSP2, CoAA Q96PK6 2 RRMs 1A SFPQ PSF P23246 2 RRMs 1A Although SR-rich splicing factors are commonly enriched in nuclear speckles, SRSF10 was exceptionally enriched in paraspeckles rather than nuclear speckles. The set of PSPs included several abundant heterogenous nuclear ribonucleoproteins (hnRNPs) (A1, A1L, F, H1, H3, K, R, and UL1). Although the numbers were limited, some of the PSPs possessed putative DNA-binding domains, including the AT-hook, Zn finger, homeodomain, and SAP domain. Several of the PSPs possessing RNA-binding domains (e.g., NONO, SFPQ, EWSR1, FUS, TAF15, and HNRNPUL1) are known to be involved in transcription. This observation suggests that the paraspeckle may serve as a platform of transcription and subsequent RNA processing events (see Discussion). Functional assignments for each PSP identified additional essential factors for paraspeckle formation To investigate the respective roles of the newly identified and known PSPs in paraspeckle construction, each PSP was knocked down with at least two independent siRNAs (Supplementary Table S2). The resultant changes in paraspeckle appearance (i.e., proportion [%] of cells possessing intact paraspeckles) and in the levels of each NEAT1 isoform (summarized in Supplementary Table S3) were examined. The PSPs were classified into three distinct categories according to the proportion of paraspeckle-possessing cells after RNAi. After treatment with control siRNA, 88% of the cells examined possessed paraspeckles; this group was defined as the control (Ctl) group (100%). Categories 1, 2, and 3 included PSPs whose RNAi led to a marked decrease (⩽30% of Ctl), substantial decrease (30–75% of Ctl), or no obvious change (⩽75% of Ctl) in the proportion of paraspeckle-possessing cells, respectively (Supplementary Table S3; Supplementary Figure S4). Investigation of the NEAT1 levels by RNase protection assays (RPAs) (Figure 3B) revealed that category 1 could be divided into two subcategories, in which the NEAT1_2 levels were markedly diminished to ⩽30% (category 1A) or were unchanged (category 1B). Similarly, category 3 was divided into two subcategories, in which NEAT1_1 levels were either diminished to ⩽30% (category 3A) or were unchanged (category 3B). The PSP categorization is summarized in Figure 4 and Table I. Representative data from each of the five categories are shown in Figure 3C–G. Figure 3.Functional assignment of new PSPs in paraspeckle formation by extensive RNAi treatment. Paraspeckle appearance and NEAT1 levels were monitored by RNA-FISH and RPA to detect NEAT1_1 and NEAT1_2. (A) Paraspeckles in cells treated with control siRNA. (B) Schema for the RPA probe used and the protected fragments (with size, nt) of NEAT1_1 and NEAT1_2. Data for a representative from each category (1A: RBM14, 1B: PSP8/DAZAP1, 2: PSP21/HNRNPR, 3A: PSP34/UBAP2L, 3B: PSP36/ZNF335) are shown in (C–G), respectively. The siRNA numbers (#) used in the RNA-FISH analysis are shown at the lower left of each photo. For RPA, the ratio of band intensities of the two isoforms, normalized by those of U12 snRNA, is shown below (Ctl was defined as 100%). RNAi data regarding all PSPs are compiled in Supplementary Figure S4. Their quantified data are shown in Supplementary Table S3. The siRNAs used are shown in Supplementary Table S6. Scale bar, 10 μm. Figure source data can be found with the Supplementary data. Supplementary Data for Figure 3 [embj2012251-sup-0002.pdf] Download figure Download PowerPoint Figure 4.Compilation of PSPs. Schematics of the major domains of the PSPs that belong to category 1 (A), 2 (B), and 3 (C) are grouped and shown. Uncategorized PSPs are shown in (D). Subcategories in categories 1 and 3 are shown as 1A and 1B in (A) and 3A and 3B in (C). The amino-acid length of each PSP is shown in the right corner. The colour codes of four putative RNA-binding domains are shown on the right. Download figure Download PowerPoint Several PSPs were unable to be categorized because their expression was either undetectable in HeLa cells (PSP5, 9, and 23) or they showed inconsistent paraspeckle phenotypes between treatments with the two siRNAs (PSP16, 17, 18, and 32). Several PSPs were tentatively categorized according to the consistent paraspeckle phenotypes between treatments with two siRNAs, although the RPA data were highly variable (PSP10, 15, 25, and 27). Three PSPs are involved in NEAT1 isoform synthesis by modulating alternative 3′-end processing Because the NEAT1 isoforms share an identical 5′-terminus, they are likely produced by alternative 3′-end processing. The 3′-ends of NEAT1_1 and NEAT1_2 are formed by two distinct mechanisms: canonical polyadenylation and RNase P cleavage, respectively. The above RNAi experiments identified factors that are involved in this alternative 3′-processing event. Two PSPs, CPSF6 and PSP24/NUDT21, form a heterodimer (CFIm complex) to facilitate the 3′-end processing of alternatively processed mRNAs (Kim et al, 2010). These PSPs also appear to act in NEAT1_1 3′-end processing. We observed that the RNAi of NUDT21 or CPSF6 markedly diminished NEAT1_1 levels and simultaneously increased the NEAT1_2 level (Figure 5A; Supplementary Figure S5B). Figure 5.Alternative 3′-end processing of NEAT1 is the initial essential step underlying paraspeckle formation. (A) PSPs required for the alternative 3′-end processing of NEAT1. RPA was performed as in Figure 3. Data are shown for NUDT21 and CPSF6, which are required for NEAT1_1 3′-end processing, and HNRNPK, which is required for NEAT1_2 synthesis by interfering with NEAT1_1 3′-end processing. (B) Paraspeckle appearance in HeLa cells treated with siRNAs against NUDT21, CPSF6, or HNRNPK. (C) Plasmid rescue of a defect of NEAT1_2 synthesis in HNRNPK-eliminated cells. Two siRNAs against HNRNPK (K#2 and K#3) were used to eliminate endogenous HNRNPK, and HNRNPK rescue plasmid (K) was introduced at three concentrations (1–5 μg). NEAT1 ncRNA levels were measured by RPA as in Figure 3. The ratios of NEAT1_1 to NEAT1_2 (NEAT1_1/NEAT1_2) are shown below the upper panel. GADPH and HNRNPK were detected by western blotting (WB). (D) Paraspeckle formation is rescued by plasmid expression of HNRNPK. The siRNAs and rescue plasmids used are shown on the left and top, respectively. Paraspeckles were detected by RNA-FISH of NEAT1. Transfected cells were visualized by immunostaining with αFlag. Arrowheads indicate paraspeckles that formed in the rescued cells. Scale bar, 10 μm. (E). Quantification of the results in (D). Cells possessing more than one paraspeckle-like focus were counted. Total cell numbers counted are the siRNAs used are shown in Supplementary Table S6. P-value was calculated by Student's t-test. The cell numbers counted for control and HNRNPK-eliminated cells were 152 and 136, respectively. Download figure Download PowerPoint The CFIm complex binds to UGUA sequences located upstream of the canonical polyadenylation signal (PAS) and recruits the general 3′-end processing machinery to polyadenylation sites (Venkataraman et al, 2005). Sequence searches revealed that five UGUA sequences are clustered 42–169 nt upstream of the PAS (AAUAAA) for NEAT1_1 3′-end processing (Figure 6A). This result strongly suggests that CFIm facilitates the 3′-end processing of NEAT1_1 through binding to the UGUA sequences. Figure 6.Roles of CFIm and HNRNPK in 3′-end processing of NEAT1_1. (A) Schematic representation of the substrate RNAs for the in vitro processing reaction that contains the region spanning the 3′-end of NEAT1_1. Numbers indicate distance from the polyadenylation site of NEAT1_1. Middle scheme represents putative sequences around the NEAT1_1 3′-end processing site that are recognized by the CFIm complex (CFBS: red boxes) or HNRNPK (KBS: blue box). Mutated positions on the mutant substrates (CFIm-mut, K-mut, and PAS-mut) are indicated. (B) Recapitulation of CFIm-dependent 3′-end processing of NEAT1_1 in vitro. Incubation time is shown above each panel. Substrate RNAs are represented on the top. Unprocessed and processed bands are shown with closed and open triangles, respectively, on the right. Processing efficiencies (%) are shown below each panel. (C) Average values of the processing efficiencies obtained from three independent experiments. (D) Detection of sequence-specific RNA binding of HNRNPK. Gel mobility shift assay to detect binding of recombinant HNRNPK protein (r-K) with RNA fragments (30 nt) derived from WT and K-mut, WT oligo, and K-mut oligo, respectively, are shown. The RNA–protein complex and free RNA are shown with closed and open triangles, respectively. Amounts of supplemented r-K (μg) are shown above each panel. Download figure Download PowerPoint Intact paraspeckles remained detectable after treatment with RNAi for either NUDT21 or CPSF6 (Figure 5B), even though NEAT1_1 was obliterated (Figure 5A; Supplementary Figure S5B). This result confirms that NEAT1_1 is dispensable for paraspeckle formation. Although RNAi elimination of PSP10/EWSR1 upregulated NEAT1_1, this condition did not affect NEAT1_2 (Supplementary Figure S4E). This observation suggests that EWSR1 may control the stability of NEAT1_1. PSP20/HNRNPK is a new member of category 1A that is required for NEAT1_2 accumulation. Treatment with HNRNPK RNAi disrupted the paraspeckles and decreased the NEAT1_2 level, but simultaneously elevated the NEAT1_1 level (>2-fold) (Figure 5A and B; Supplementary Figure S5B). This finding was not observed with an RNAi of any other category I protein (Supplementary Figure S4A), which suggests that HNRNPK facilitates NEAT1_2 synthesis, rather than stabilization, by modulating NEAT1_1 3′-end processing. RT–qPCR measurement of NEAT1 ncRNA coimmunoprecipitated with anti-NUDT21 antib
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