Jeongsik Yong

RNA-binding proteins and post-transcriptional gene regulation.

RNAs in cells are associated with RNA‐binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization. Eukaryotic cells encode a large number of RBPs (thousands in vertebrates), each of which has unique RNA‐binding activity and protein–protein interaction characteristics. The remarkable diversity of RBPs, which appears to have increased during evolution in parallel to the increase in the number of introns, allows eukaryotic cells to utilize them in an enormous array of combinations giving rise to a unique RNP for each RNA. In this short review, we focus on the RBPs that interact with pre‐mRNAs and mRNAs and discuss their roles in the regulation of post‐transcriptional gene expression.

Pre-mRNA Splicing Imprints mRNA in the Nucleus with a Novel RNA-Binding Protein that Persists in the Cytoplasm

We describe a novel RNA binding protein, Y14, a predominantly nuclear nucleocytoplasmic shuttling protein. Interestingly, Y14 associates preferentially with mRNAs produced by splicing but not with pre-mRNAs, introns, or mRNAs produced from intronless cDNAs. Y14 associates with both nuclear mRNAs and newly exported cytoplasmic mRNAs. Splicing of a single intron is sufficient for Y14 association. Y14-containing nuclear complexes are different from general hnRNP complexes. They contain hnRNP proteins and several unique proteins including the mRNA export factor TAP. Thus, Y14 defines novel intermediates in the pathway of gene expression, postsplicing nuclear preexport mRNPs, and newly exported cytoplasmic mRNPs, whose composition is established by splicing. These findings suggest that pre-mRNA splicing imprints mRNA with a unique set of proteins that persists in the cytoplasm and thereby communicates the history of the transcript.

SMN interacts with a novel family of hnRNP and spliceosomal proteins

Spinal muscular atrophy (SMA) is a common neurodegenerative disease caused by deletion or loss‐of‐function mutations of the survival of motor neurons (SMN) protein. SMN is in a complex with several proteins, including Gemin2, Gemin3 and Gemin4, and it plays important roles in small nuclear ribonucleoprotein (snRNP) biogenesis and in pre‐mRNA splicing. Here, we characterize three new hnRNP proteins, collectively referred to as hnRNP Qs, which are derived from alternative splicing of a single gene. The hnRNP Q proteins interact with SMN, and the most common SMN mutant found in SMA patients is defective in its interactions with them. We further demonstrate that hnRNP Qs are required for efficient pre‐mRNA splicing in vitro. The hnRNP Q proteins may provide a molecular link between the SMN complex and splicing.

Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing‐dependent exon–exon junction complex

The RNA‐binding protein Y14 binds preferentially to mRNAs produced by splicing and is a component of a multiprotein complex that assembles ∼20 nucleotides upstream of exon–exon junctions. This complex probably has important functions in post‐splicing events including nuclear export and nonsense‐mediated decay of mRNA. We show that Y14 binds to two previously reported components, Aly/REF and RNPS1, and to the mRNA export factor TAP. Moreover, we identified magoh, a human homolog of the Drosophila mago nashi gene product, as a novel component of the complex. Magoh binds avidly and directly to Y14 and TAP, but not to other known components of the complex, and is found in Y14‐containing mRNPs in vivo. Importantly, magoh also binds to mRNAs produced by splicing upstream (∼20 nucleotides) of exon– exon junctions and its binding to mRNA persists after export. These experiments thus reveal specific protein–protein interactions among the proteins of the splicing‐dependent mRNP complex and suggest an important role for the highly evolutionarily conserved magoh protein in this complex.

The Y14 protein communicates to the cytoplasm the position of exon–exon junctions

We recently described an RNA‐binding protein, Y14, that binds preferentially to spliced mRNAs and persists in the cytoplasm. Y14 is part of a multi‐protein complex that also contains the mRNA export factor TAP. This suggests that splicing imprints the mRNA with a unique set of proteins that communicate the history of the transcript to the cytoplasm. Here, using microinjection of pre‐mRNAs into Xenopus oocyte nuclei followed by immunoprecipitation of RNase‐fragmented mRNAs from the cytoplasm, we show that Y14 is stably bound to sequences immediately upstream of exon–exon junctions. This feature appears to be unique to Y14. Using monoclonal antibodies that we produced against Aly/REF, another component recently reported to be an mRNA export factor, we show that Aly/REF is associated with spliced mRNAs in the nucleus but is not detectable on mRNAs in the cytoplasm. Thus, we propose that the splicing‐ dependent binding of Y14 provides a position‐specific molecular memory that communicates to the cytoplasm the location of exon and intron boundaries. This novel mechanism may play an important role in post‐splicing events.

The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

ABSTRACT Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

tRNA Binds to Cytochrome c and Inhibits Caspase Activation

The specific molecular events that characterize the intrinsic apoptosis pathway have been the subject of intense research due to the pathways fundamental role in development, homeostasis, and cancer. This pathway is defined by the release of cytochrome c from mitochondria into the cytosol and subsequent binding of cytochrome c to the caspase activator Apaf-1. Here, we report that both mitochondrial and cytosolic transfer RNA (tRNA) bind to cytochrome c. This binding prevents cytochrome c interaction with Apaf-1, blocking Apaf-1 oligomerization and caspase activation. tRNA hydrolysis in living cells and cell lysates enhances apoptosis and caspase activation, whereas microinjection of tRNA into living cells blocks apoptosis. These findings suggest that tRNA, in addition to its well-established role in gene expression, may determine cellular responsiveness to apoptotic stimuli.

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem–loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence‐specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.

snRNAs Contain Specific SMN-Binding Domains That Are Essential for snRNP Assembly

ABSTRACT To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.