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Post-transcriptional modification of RNA nucleosides has been implicated as a pivotal regulator of mRNA biology. In this issue of Genes & Development , Ke and colleagues (pp. 990-1006) provide insights into the temporal and spatial distribution of N 6 -methyladenosine (m 6 A) in RNA transcripts by analyzing different subcellular fractions. Using a recently developed biochemical approach for detecting m 6 A, the researchers show that m 6 A methylations are enriched in exons and are added to transcripts prior to splicing. Although m 6 A addition is widely thought to be readily reversible, they demonstrate in HeLa cells that once RNA is released from chromatin, the modifications are surprisingly static. This study integrates data from previous publications to clarify conflicting conclusions regarding the role of m 6 A in mRNA biogenesis and function. Ke and colleagues found that m 6 A methylation levels negatively correlate with transcript half-life but are not required for most pre-mRNA splicing events., (© 2017 Rosa-Mercado et al.; Published by Cold Spring Harbor Laboratory Press.)

Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology., (© 2015 Xie et al.; Published by Cold Spring Harbor Laboratory Press.)

Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles--including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation--have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action., (© 2015 Tycowski et al.; Published by Cold Spring Harbor Laboratory Press.)

Despite the lack of an exon junction complex (EJC), Saccharomyces cerevisiae contains Fal1p, a DEAD-box helicase highly homologous to eIF4AIII. We show that yeast Fal1p is functionally orthologous to human eIF4AIII, since expression of human eIF4AIII complements both the lethal phenotype and the 18S rRNA biogenesis defect of fal1Δ(null) yeast. We further show that yeast Fal1p interacts genetically with an eIF4G-like protein, Sgd1p: One allele of sgd1 acts as a dominant extragenic suppressor of a mutation in a predicted RNA-binding residue of Fal1p, whereas another synthetically exacerbates the growth defect of this fal1 mutation. Both sgd1 mutations map to a single, short, evolutionarily conserved patch that matches key eIF4A-interacting residues of eIF4G when superimposed on the X-ray structure of the eIF4A/eIF4G complex. We demonstrate direct physical interactions between yeast Sgd1p and Fal1p, and between their human orthologs (NOM1 and eIF4AIII) in vitro and in vivo, identifying human NOM1 as a missing eIF4G-like interacting partner of eIF4AIII. Knockdown of eIF4AIII and NOM1 in human cells demonstrates that this novel conserved eIF4A/eIF4G-like complex acts in pre-rRNA processing, adding to the established functions of eIF4A/eIF4G in translation initiation and of eIF4AIII as the core component of the EJC.

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation.

An in vitro system that recapitulates the in vivo effect of AU-rich elements (AREs) on mRNA deadenylation has been developed from Xenopus activated egg extracts. ARE-mediated deadenylation is uncoupled from mRNA body decay, and the rate of deadenylation increases with the number of tandem AUUUAs. A novel ARE-binding protein called ePAB (for embryonic poly(A)-binding protein) has been purified from this extract by ARE affinity selection. ePAB exhibits 72% identity to mammalian and Xenopus PABP1 and is the predominant poly(A)-binding protein expressed in the stage VI oocyte and during Xenopus early development. Immunodepletion of ePAB increases the rate of both ARE-mediated and default deadenylation in vitro. In contrast, addition of even a small excess of ePAB inhibits deadenylation, demonstrating that the ePAB concentration is critical for determining the rate of ARE-mediated deadenylation. These data argue that ePAB is the poly(A)-binding protein responsible for stabilization of poly(A) tails and is thus a potential regulator of mRNA deadenylation and translation during early development.

We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome.

AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3'-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA.

SR proteins are essential splicing factors that also influence 5' splice site choice. We show that addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection.

We have generated mutants in Xenopus U8 RNA, a nucleolar snRNA required for the maturation of 5.8S and 28S rRNAs, to identify sequences and structural domains essential for RNA stability, particle assembly, and function of the U8 RNP. Activity of the mutants was assayed by microinjection of in vitro-synthesized U8 RNAs into the cytoplasm of Xenopus oocytes. Most of the mutant RNAs were stable, bound fibrillarin, a protein common to several of the nucleolar-specific snRNPs, and became hypermethylated. Although hypermethylation of the 5' cap of U8 RNA and fibrillarin binding can occur in either the cytoplasmic or nuclear compartment of Xenopus oocytes, neither is required for nuclear import. We find that the trimethylguanosine cap, although present on the endogenous U8 RNA, is not essential for stability, particle assembly, or functioning of U8 in the coordinate processing of pre-rRNA at sites 3' of 28S and 5' of 5.8S RNA. Several conserved single- and double-stranded sequences within the 5' domain of U8 RNA are essential for function.

A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides), which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein S3 gene; the U15A sequence appears on the same strand as the S3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from S3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells.

Psoralen cross-linking experiments in HeLa cell nuclear extracts have revealed the binding of U1 snRNA to substrates containing the SV40 late and adenovirus L3 polyadenylation signals. The sites of U1 cross-linking to the substrates map different distances upstream of the AAUAAA sequence to regions with limited complementarity to the 5' end of U1 snRNA. U1 cross-linking to the same site in the SV40 late pre-mRNA is enhanced by the addition of an upstream 3' splice site, which also enhances polyadenylation. Examination of different nuclear extracts reveals a correlation between U1 cross-linking and the coupling of splicing and polyadenylation, suggesting that the U1 snRNP participates in the coordination of these two RNA-processing events. Mutational analyses demonstrate that U1/substrate association cannot be too strong for coupling to occur and suggest that the U1 snRNP plays a similar role in recognition of internal and 3' terminal exons. Possible mechanisms for communication between the splicing and polyadenylation machineries are discussed, as well as how interaction of the U1 snRNP with 3' terminal exons might contribute to mRNA export.

We have used a site-specific cross-linking strategy to identify RNA and protein factors that interact with the 5' splice site region during mammalian pre-mRNA splicing. Two different pre-mRNA substrates were synthesized with a single 32P-labeled 4-thiouridine residue 2 nucleotides upstream of the 5' splice site. Selective photoactivation of the 4-thiouridine residue after incubation of either substrate under splicing conditions in HeLa nuclear extract resulted in cross-links to the U5 snRNA and the U5 snRNP protein p220. These ATP-dependent interactions occur before the first step of splicing. The U5 snRNA cross-links map to a phylogenetically invariant 9-nucleotide loop sequence and do not require Watson-Crick complementarity to the 5' exon. Cross-links of this position in the pre-mRNA to U1, but not to U2, U4, or U6 snRNAs, were also observed. The kinetics of U1 and U5 cross-link formation are similar, both peaking well before reaction intermediates appear.

To study the role of base-pairing between the mammalian U7 snRNA and the highly variable histone downstream element (HDE) during the 3'-end maturation of mammalian histone pre-mRNAs, we mutated the HDE of the mouse H2A-614 gene and assayed processing in HeLa cells both in vivo and in vitro. Either a 9-nucleotide deletion or a block substitution of pyrimidines for 6 purines within the HDE abolished all 3'-end processing. Compensatory changes were introduced into a synthetic human U7 gene, whose transcripts assemble into Sm snRNPs in vivo. Suppression of the 6-purine substitution as well as a 3-purine substitution within the HDE was obtained in vivo by coexpressing the corresponding U7 suppressor RNAs and in vitro by using nuclear extracts prepared from HeLa cells containing U7 suppressor genes. Our results not only provide genetic evidence for base-pairing between the U7 snRNP and the HDE of mammalian histone pre-mRNAs but reveal an unexpected tolerance to drastic changes in the nature of the base-paired region.

In transcripts from the rearranged mouse immunoglobulin kappa light-chain locus, the intron separating the variable (V) plus joining (J) exon from the constant (C) exon contains up to three additional J regions, each with a functional 5' splice site. Previously, HeLa cells transfected with DNA encoding kappa light chains have been shown to mimic kappa-producing lymphocytes in splicing exclusively to the upstream-most 5' splice site, whereas selectivity is lost when kappa transcripts containing two more J regions are incubated in HeLa cell or lymphocyte nuclear extracts. Here we demonstrate that the fidelity of in vivo splicing depends on neither V-J rearrangement, the instability of erroneously splicing transcripts, nor a hierarchy of J-region 5' splice site utilization. Analysis of the splicing of presynthesized kappa transcripts injected into Xenopus oocytes demonstrates the correct 5' splice-site selection is independent of transcription. Implications for in vitro studies of regulated splice-site pairing are discussed.