SUMMARYPUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar yet distinct mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode and mammalian PUF/Ago/eEF1A complexes were identified, using co-immunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promotes repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1/CSR-1 heterodimer inhibits EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2/Ago/eEF1A inhibits translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurs after translation initiation and leads to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerges from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF/Ago/eEF1A complex attenuates translation elongation.

The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA.

In metazoa, pre-mRNA 3' end formation occurs via two pathways: cleavage/polyadenylation for the majority of RNA polymerase II transcripts and U7-snRNP-dependent cleavage for replication-dependent histone pre-mRNAs. An RNA element derived from a replication-dependent histone gene affects multiple steps of pre-mRNA processing. Here, we demonstrate that a portion of this RNA element, present in the majority of histone mRNAs, stimulates U7-snRNP-dependent cleavage. Surprisingly, this element binds U2 snRNP, although it is derived from an intronless mRNA. Specifically, SF3b, a U2 and U12-snRNP component, contacts the RNA element both in vitro and in vivo in conjunction with hPrp43, a DEAH-box helicase. Tethering either U2 or U12 snRNP to histone pre-mRNA substrates stimulates U7-snRNP-dependent cleavage in vitro and in vivo. Finally, we show that U2 snRNP associates with histone pre-mRNAs in vivo. We conclude that U2 snRNP plays a nonsplicing role in histone mRNA maturation.