Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles

Abstract

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.

SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies

“…HNRNPK, NONO, RBM14, and SFPQ (category 1A proteins), but not DAZAP1, FUS, or HNRNPH3 (category 1B proteins), are required for the accumulation of the essential NEAT1_2 isoform (14). Whereas HNRNPK facilitates NEAT1_2 synthesis by interfering with the 3′-end processing of NEAT1_1 (14), other proteins in category 1A stabilize the NEAT1_2 isoform. Although category 1B proteins barely affect NEAT1_2 accumulation, they are required for the assembly of intact paraspeckles (14).…”

“…The NEAT1 gene generates two isoform transcripts, namely, 3.7-kb NEAT1_1 and 23-kb NEAT1_2. Both isoforms are transcribed from the same promoter and can be processed at the 3′ end to produce a canonically polyadenylated NEAT1_1 isoform and a noncanonically processed NEAT1_2 isoform (6,7,14). Whereas NEAT1_2 is required for de novo paraspeckle construction, NEAT1_1 is not required for this process (6,14,15).…”

“…Whereas NEAT1_2 is required for de novo paraspeckle construction, NEAT1_1 is not required for this process (6,14,15). Extensive RNAi analyses of 40 paraspeckle proteins (PSPs) revealed that seven PSPs, namely heterogeneous nuclear RNP K (HNRNPK), NONO, RNAbinding motif protein 14 (RBM14), SFPQ, DAZ-associated protein 1 (DAZAP1), fused in sarcoma (FUS), and HNRNPH3, are essential for paraspeckle formation (14). HNRNPK, NONO, RBM14, and SFPQ (category 1A proteins), but not DAZAP1, FUS, or HNRNPH3 (category 1B proteins), are required for the accumulation of the essential NEAT1_2 isoform (14).…”

“…Both isoforms are transcribed from the same promoter and can be processed at the 3′ end to produce a canonically polyadenylated NEAT1_1 isoform and a noncanonically processed NEAT1_2 isoform (6,7,14). Whereas NEAT1_2 is required for de novo paraspeckle construction, NEAT1_1 is not required for this process (6,14,15). Extensive RNAi analyses of 40 paraspeckle proteins (PSPs) revealed that seven PSPs, namely heterogeneous nuclear RNP K (HNRNPK), NONO, RNAbinding motif protein 14 (RBM14), SFPQ, DAZ-associated protein 1 (DAZAP1), fused in sarcoma (FUS), and HNRNPH3, are essential for paraspeckle formation (14).…”

“…Extensive RNAi analyses of 40 paraspeckle proteins (PSPs) revealed that seven PSPs, namely heterogeneous nuclear RNP K (HNRNPK), NONO, RNAbinding motif protein 14 (RBM14), SFPQ, DAZ-associated protein 1 (DAZAP1), fused in sarcoma (FUS), and HNRNPH3, are essential for paraspeckle formation (14). HNRNPK, NONO, RBM14, and SFPQ (category 1A proteins), but not DAZAP1, FUS, or HNRNPH3 (category 1B proteins), are required for the accumulation of the essential NEAT1_2 isoform (14). Whereas HNRNPK facilitates NEAT1_2 synthesis by interfering with the 3′-end processing of NEAT1_1 (14), other proteins in category 1A stabilize the NEAT1_2 isoform.…”