Ichiro Taniguchi

hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export.

Choosing the Right Path RNA molecules are synthesized in the cell nucleus, yet many have to be moved to the cytoplasm to be processed and/or to effect their function. Different classes of RNA are transported from the nucleus by different transport systems. Messenger RNAs (mRNAs) and uridine-rich small nuclear RNAs (U snRNAs) are transcribed by RNA polymerase II and are capped and bound by the cap-binding machinery in the nucleus but are exported by different protein complexes. The feature that distinguishes the two classes of RNA is their length: U snRNAs are short and mRNAs are long. Using an in vitro system and human tissue culture cells, McCloskey et al. (p. 1643) show that the length of the RNAs is measured by the heterogeneous nuclear ribonicleoprotein (hnRNP) C tetrameric protein complex. The hnRNP C cannot bind to the short U snRNAs, allowing the U snRNA-specific export adaptor protein, PHAX, to bind and mediate export. Longer mRNAs are bound by hnRNP C, which prevents the binding of PHAX, thus identifying these RNAs for export from the nucleus via the mRNA pathway. A nuclear protein measures the length of newly made RNAs and sorts them into distinct pathways for export. Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56

ABSTRACT Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells

Intron‐containing pre‐mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre‐mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre‐mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre‐mRNAs in mammalian cells. By focusing on factors that bind to the 3′‐splice site region, we found that the 65‐kD subunit of U2AF (U2AF65) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD‐box RNA helicase involved in both RNA splicing and export, cooperates with U2AF65 in exerting nuclear retention activity. Our findings provide new information regarding the pre‐mRNA nuclear retention factors in mammalian cells.

Role of purine-rich exonic splicing enhancers in nuclear retention of pre-mRNAs

Intron-containing pre-mRNAs are normally retained in the nucleus until they are spliced to produce mature mRNAs that are exported to the cytoplasm. Although the detailed mechanism is not well understood, the formation of splicing-related complexes on pre-mRNAs is thought to be responsible for the nuclear retention. Therefore, pre-mRNAs containing suboptimal splice sites should tend to leak out to the cytoplasm. Such pre-mRNAs often contain purine-rich exonic splicing enhancers (ESEs) that stimulate splicing of the adjacent intron. Here, we show that ESEs per se possess an activity to retain RNAs in the nucleus through a saturable nuclear retention factor. Cross-competition experiments revealed that intron-containing pre-mRNAs (without ESEs) used the same saturable nuclear retention factor as ESEs. Interestingly, although intronless mRNAs containing ESEs were also poorly exported, spliced mRNAs produced from ESE-containing pre-mRNAs were efficiently exported to the cytoplasm. Thus, the splicing reaction can reset the nuclear retention state caused by ESEs, allowing nuclear export of mature mRNAs. Our results reveal a novel aspect of ESE activity that should contribute to gene expression and RNA quality control.

U1-independent pre-mRNA splicing contributes to the regulation of alternative splicing.

U1 snRNP plays a crucial role in the 5′ splice site recognition during splicing. Here we report the first example of naturally occurring U1-independent U2-type splicing in humans. The U1 components were not included in the pre-spliceosomal E complex formed on the human F1γ (hF1γ) intron 9 in vitro. Moreover, hF1γ intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts. Finally, hF1γ exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one. Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV‐crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine‐rich) proteins, a family of essential RNA‐binding proteins involved in pre‐mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre‐mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

Exportin‐5 mediates nuclear export of SRP RNA in vertebrates

The signal recognition particle is a ribonucleoprotein complex that is essential for the translocation of nascent proteins into the endoplasmic reticulum. It has been shown that the RNA component (SRP RNA) is exported from the nucleus by CRM1 in the budding yeast. However, how SRP RNA is exported in higher species has been elusive. Here, we show that SRP RNA does not use the CRM1 pathway in Xenopus oocytes. Instead, SRP RNA uses the same export pathway as pre‐miRNA and tRNA as showed by cross‐competition experiments. Consistently, the recombinant Exportin‐5 protein specifically stimulated export of SRP RNA as well as of pre‐miRNA and tRNA, whereas an antibody raised against Exportin‐5 specifically inhibited export of the same RNA species. Moreover, biotinylated SRP RNA can pull down Exportin‐5 but not CRM1 from HeLa cell nuclear extracts in a RanGTP‐dependent manner. These results, taken together, strongly suggest that the principal export receptor for SRP RNA in vertebrates is Exportin‐5 unlike in the budding yeast.

Analysis of RNA Transport in Xenopus Oocytes and Mammalian Cells

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.