James E. Dahlberg

The constitutive transport element (CTE) of Mason–Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway

The constitutive transport elements (CTEs) of type D retroviruses are cis‐acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV‐1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason–Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV‐1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE‐containing lariat occurs when wild‐type CTE, but not a mutant form, is inserted into the pre‐mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non‐functional variants, making these proteins candidates for the sequestered mRNA export factors.

The Genes and Transcription of the Major Small Nuclear RNAs

Several unusual features distinguish snRNA genes and make snRNA synthesis an important and interesting subject for study. Although the snRNA genes are very efficiently and accurately transcribed by transcription complexes which use RNA polymerase II (or III, in the case of U6 genes), these genes contain transcription signals that differ from those normally recognized by RNA polymerase II.

The matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes.

ABSTRACT The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of bidirectional nuclear transport. Here we demonstrate that inhibition occurs when M protein is in the nucleus ofXenopus laevis oocytes and that M activity is readily reversed by a monoclonal antibody (αM). We identify a region of M protein, amino acids 51 to 59, that is required both for inhibition of transport and for efficient recognition by αM. When expressed in transfected HeLa cells, M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim. Moreover, mutation of a single amino acid, methionine 51, eliminates both transport inhibition and targeting to NPCs. We propose that M protein inhibits bidirectional transport by interacting with a component of the NPC or an NPC-associated factor that participates in nucleocytoplasmic transport.

Nuclear export of ribosomal subunits

The partitioning of cells by a nuclear envelope ensures that precursors of ribosomes do not interact prematurely with other components of the translation machinery. Ribosomal subunits are assembled in nucleoli and exported to the cytoplasm in a CRM1/Ran-GTP-dependent fashion. Export of the large (60S) subunit requires a shuttling adaptor protein, NMD3, which binds to mature, correctly folded subunits. Immature or defective particles do not bind NMD3 and thus are excluded from the export pathway. This structural proofreading is extended into the cytoplasm, where it is believed that several energy-requiring steps release shuttling factors from the subunit, allowing it to function in translation.

Transfer RNA genes between 16S and 23S rRNA genes in rRNA transcription units of E. Coli

We have identified genes for tRNAGLU/2 on the transducing phages o80d3ilvsu7+ (see Ohtsubo et al., 1974) and lambdarifd18 (Kirschbaum and Konrad, 1973), and a gene for tRNAlle/1 on the transducing phage o80rifr (Konrad, Kirschbaum, and Austin, 1973). All these phages have previously been shown to carry genes for rRNA (Ohtsubo et al., 1974; Lindahl et al., 1975; Jaskunas et al., 1975a). We have analyzed the position of these tRNA genes by hybridizing purified RNAs to restriction fragments of the phage DNA. The tRNA genes are located inside the rRNA transcription unit in the spacer region between the 16S and 23S rRNA genes.

miR-155/BIC as an oncogenic microRNA.

A group of recently published articles on microRNAs (miRNAs; He et al., 2005; Lu et al., 2005; O’Donnell et al., 2005) supports arguments that noncoding RNAs play important roles in oncogenesis and hence may be valuable as diagnostic markers and therapeutic targets. To our knowledge, the first miRNA-encoding RNA shown to participate in oncogenesis was BIC RNA, which was originally identified several years ago as a noncoding RNA from a common retroviral integration site in avian leukosis virus–induced lymphomas (Tam et al., 1997; McManus, 2003). It was subsequently shown that overexpression of a highly structured, phylogenetically conserved region in the last exon of BIC RNA led to an increase in the incidence of leukemia and a decrease in the latency of lymphoma development in chickens with elevated levels of MYC (Tam et al., 2002). This work was done before the dawn of the miRNA era; despite recognition of the potential significance of the highly ordered RNA structure (Tam, 2001), the mechanism of oncogenicity of this noncoding RNA remained a mystery until recently. We now understand that this region encompasses the pre-miR155 sequence and that elevated levels of BIC RNA are associated with elevated levels of miR-155 (Eis et al., 2005; Kluiver et al., 2005). It follows that miR-155 is very likely to be a contributing factor in the etiology of human lymphomas. Our recent work has shown that in certain human diffuse large B-cell lymphomas, the levels of miR155 increase as much as 60-fold above normal and that higher levels are associated with poorer clinical prognosis (Eis et al., 2005). BIC/miR-155 is also elevated in Hodgkin lymphoma cell lines and clinical samples (van den Berg et al., 2003; Eis et al., 2005; Kluiver et al., 2005), as well as in childhood Burkitt’s lymphoma (Metzler et al., 2004). To our knowledge, the work on BIC and MYC in a chicken lymphoma model represents the first example of an miRNA-encoding gene shown directly to function as an oncogene. Despite its similarity to the oncogenic collaboration between miR-17-92 and MYC demonstrated in mice (He et al., 2005), our earlier work was not mentioned in the article by He at al., and we think it deserves the recognition. It remains to be seen if miRNAs derived from the miR-17-92 polycistron and miR155 affect the same or different oncogenic pathways. Regardless, miR-155 should also be considered an onco-miRNA. The mechanisms by which these onco-miRNAs contribute to tumor initiation and progression will be a major focus of cancer research for years to come.

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran·GTP. Export of 60S subunits is coupled with that of the nucleo‐ cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM‐1‐dependent leucine‐rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild‐type and export‐defective mutant hNMD3 proteins bind to newly made nuclear 60S pre‐export particles at a late step of subunit maturation. The export‐defective hNMD3, but not the wild‐type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild‐type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1–Ran·GTP‐mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.

A common maturation pathway for small nucleolar RNAs.

We have shown that precursors of U3, U8 and U14 small nucleolar RNAs (snoRNAs) are not exported to the cytoplasm after injection into Xenopus oocyte nuclei but are selectively retained and matured in the nucleus, where they function in pre‐rRNA processing. Our results demonstrate that Box D, a conserved sequence element found in these and most other snoRNAs, plays a key role in their nuclear retention, 5′ cap hypermethylation and stability. Retention of U3 and U8 RNAs in the nucleus is saturable and relies on one or more common factors. Hypermethylation of the 5′ caps of U3 RNA occurs efficiently in oocyte nuclear extracts lacking nucleoli, suggesting that precursor snoRNAs are matured in the nucleoplasm before they are localized to the nucleolus. Surprisingly, m7G‐capped precursors of spliceosomal small nuclear RNAs (snRNAs) such as pre‐U1 and U2, can be hypermethylated in nuclei if the RNAs are complexed with Sm proteins. This raises the possibility that a single nuclear hypermethylase activity may act on both nucleolar and spliceosomal snRNPs.

Differential expression of multiple U1 small nuclear RNAs in oocytes and embryos of Xenopus laevis

The small nuclear RNA, U1, is a highly conserved, 165 nucleotide long RNA which has been implicated in the processing of mRNA precursors. We present evidence that in the amphibian X. laevis there exist at least seven species of U1 RNA, which differ in sequence but not in length. Strikingly, these RNAs are not coordinately expressed. Two of the U1 RNAs are the predominant U1 species transcribed in the late blastula-early gastrula stages of Xenopus embryogenesis. These two RNAs, designated xU1a and xU1b, are not synthesized in significant amounts in stage 6 oocytes; a different set of U1 RNAs are expressed during late oogenesis. In a Xenopus cultured cell line, all of the U1 RNA species are expressed. Possible functions and developmental significance of these multiple U1 RNA species are discussed.

Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in Xenopus laevis

We show that, in Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics in either deadenylation or guided mRNA cleavage (RNAi). Instead, siRNAs saturate and inactivate maternal Argonaute (Ago) proteins, which are present in low amounts but are needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, siRNAs impair accumulation of newly made miRNAs, such as the abundant embryonic pre-miR-427, but inhibition dissipates upon synthesis of zygotic Ago proteins after MBT. These effects of siRNAs, which are independent of sequence, result in morphological defects at later stages of development. The expression of any of several exogenous human Ago proteins, including catalytically inactive Ago2 (Ago2mut), can overcome the siRNA-mediated inhibition of miR-427 biogenesis and function. However, expression of wild-type, catalytically active hAgo2 is required to elicit RNAi in both early embryos and oocytes using either siRNA or endogenous miRNAs as guides. The lack of endogenous Ago2 endonuclease activity explains why these cells normally are unable to support RNAi. Expression of catalytically active exogenous Ago2, which appears not to perturb normal Xenopus embryonic development, can now be exploited for RNAi in this vertebrate model organism.