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Joan A Steitz

Publications and source records attributed to Joan A Steitz.

At least 19 recordsLinked to original sources
PubMed2006

Identification of a rapid mammalian deadenylation-dependent decay pathway and its inhibition by a viral RNA element.

Cellular RNAs are subject to quality-control pathways that insure the fidelity of gene expression. We previously identified a 79 nt element, the ENE, that is essential for the nuclear accumulation of a viral polyadenylated nuclear (PAN) RNA. Here, we show that intron-less polyadenylated transcripts such as PAN RNA and beta-globin cRNA exhibit two-component exponential decay kinetics in which some transcripts are rapidly degraded (t(1/2) = approximately 15 min) while others decay more slowly (t(1/2) = approximately 3 hr). Inclusion of the ENE protects such transcripts from rapid decay in a poly(A)-dependent fashion. The ENE inhibits deadenylation and decay in nuclear extract and prevents deadenylation of naked RNA by a purified deadenylase, likely through snoRNA-like intramolecular hybridization with the poly(A) tail. The ENE causes increased accumulation of splicing-defective beta-globin pre-mRNAs in vivo. We propose that the ENE-controlled rapid-decay mechanism for polyadenylated transcripts comprises a nuclear pre-mRNA surveillance system in mammalian cells.

Base Sequence↗
PubMed2006

A spliceosomal intron binding protein, IBP160, links position-dependent assembly of intron-encoded box C/D snoRNP to pre-mRNA splicing.

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.

Animals↗
PubMed2006

Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling.

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.

Active Transport, Cell Nucleus↗
PubMed2006

Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22.

EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.

Binding Sites↗
PubMed2006

In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site.

Most histone precursor mRNAs (pre-mRNAs) in metazoans are matured by 3'-end cleavage directed by the U7 small nuclear ribonucleoprotein (snRNP). RNA functional groups necessary for in vivo assembly and activity of the U7 snRNP were examined by nucleotide-analog interference mapping and mutagenesis using a chimeric mouse histone H4 pre-mRNA-U7 snRNA construct that is cleaved in cis in Xenopus laevis oocytes. Assembly of the unique U7 Sm protein core is rate limiting for processing in vivo and requires four conserved nucleotides within the U7 Sm-binding site, as well as the correct positioning and size of the U7 terminal stem-loop structure. To our surprise, pseudouridine substitution revealed a requirement for backbone flexibility at a particular position within the U7 Sm site, providing in vivo biochemical evidence that an unusual C2'-endo sugar conformation is necessary for assembly of the Sm ring.

Animals↗
PubMed2005

Symplekin and multiple other polyadenylation factors participate in 3'-end maturation of histone mRNAs.

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.

Animals↗
PubMed2005

Small nuclear RNAs encoded by Herpesvirus saimiri upregulate the expression of genes linked to T cell activation in virally transformed T cells.

Seven small nuclear RNAs of the Sm class are encoded by Herpesvirus saimiri (HVS), a gamma Herpesvirus that causes aggressive T cell leukemias and lymphomas in New World primates and efficiently transforms T cells in vitro. The Herpesvirus saimiri U RNAs (HSURs) are the most abundant viral transcripts in HVS-transformed, latently infected T cells but are not required for viral replication or transformation in vitro. We have compared marmoset T cells transformed with wild-type or a mutant HVS lacking the most highly conserved HSURs, HSURs 1 and 2. Microarray and Northern analyses reveal that HSUR 1 and 2 expression correlates with significant increases in a small number of host mRNAs, including the T cell-receptor beta and gamma chains, the T cell and natural killer (NK) cell-surface receptors CD52 and DAP10, and intracellular proteins--SKAP55, granulysin, and NKG7--linked to T cell and NK cell activation. Upregulation of three of these transcripts was rescued after transduction of deletion-mutant-HVS-transformed cells with a lentiviral vector carrying HSURs 1 and 2. These changes indicate an unexpected role for the HSURs in regulating a remarkably defined and physiologically relevant set of host targets involved in the activation of virally transformed T cells during latency.

Animals↗
PubMed2005

A Kaposi's sarcoma virus RNA element that increases the nuclear abundance of intronless transcripts.

The Kaposi's sarcoma-associated herpesvirus produces a 1077 nucleotide noncoding, polyadenylated, exclusively nuclear RNA called PAN that is highly expressed in lytically infected cells. We report that PAN contains a novel post-transcriptional element essential for its abundant accumulation. The element, PAN-ENE (PAN RNA expression and nuclear retention element), increases the efficiency of 3'-end formation in vivo and is sufficient to enhance RNA abundance from an otherwise inefficiently expressed intronless beta-globin construct. The PAN-ENE does not concomitantly increase the production of encoded protein. Rather, it retains the unspliced beta-globin mRNA in the nucleus. Tethering of export factors can override the nuclear retention of the PAN-ENE, supporting a mechanism whereby the PAN-ENE blocks assembly of an export-competent mRNP. The activities of the PAN-ENE are specific to intronless constructs, since inserting the PAN-ENE into a spliced beta-globin construct has no effect on mRNA abundance and does not affect localization. This is the first characterization of a cis-acting element that increases RNA abundance of intronless transcripts but inhibits assembly of an export-competent mRNP.

Base Sequence↗
PubMed2005

SRprises along a messenger's journey.

SR (serine/arginine-rich) proteins were originally identified as essential splicing factors. Recent work, however, has implicated these proteins in numerous additional steps of mRNA metabolism, including nuclear export, RNA stability, mRNA quality control, and translation.

Animals↗
PubMed2005

An embryonic poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early preimplantation embryos.

Gene expression during oocyte maturation, fertilization, and early embryo development until zygotic gene activation is regulated mainly by translational activation of maternally derived mRNAs. This process requires the presence of a poly(A)-binding protein. However, the cytoplasmic somatic cell poly(A)-binding protein (PABP1) is not expressed until later in embryogenesis. We recently identified an embryonic poly(A)-binding protein (ePAB) in Xenopus. ePAB is the predominant cytoplasmic PABP in Xenopus oocytes and early embryos and prevents deadenylation of mRNAs, suggesting its importance in the regulation of gene expression during early Xenopus development. Here we report the identification of the mouse ortholog of Xenopus ePAB. The mouse ePAB gene on chromosome 2 contains 14 exons that specify an alternatively spliced mRNA encoding a protein of 608 or 561 aa with approximately 65% identity to Xenopus ePAB. Mouse ePAB mRNA is expressed in ovaries and testis but not in somatic tissues. In situ hybridization localizes ePAB RNA to oocytes and confirms its absence from surrounding somatic cells in the mouse ovary. During early development, mouse ePAB is expressed in prophase I and metaphase II oocytes and one-cell and two-cell embryos and then becomes undetectable in four-or-more-cell embryos. In contrast, PABP1 mRNA expression is minimal in oocytes and early embryos until the eight-cell stage when it increases, becoming predominant at the blastocyst stage. The expression of mouse ePAB before zygotic gene activation argues for its importance in translational activation of maternally derived mRNAs during mammalian oocyte and early preimplantation embryo development.

Amino Acid Sequence↗
PubMed2005

Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein.

The interaction between box C/D small nucleolar (sno)RNAs and the 15.5K protein nucleates snoRNP assembly. Many eukaryotic snoRNAs contain two potential binding sites for this protein, only one of which appears to be utilized in vivo. The binding site conforms to the consensus for a kink-turn motif. We have investigated the molecular basis for selection of one potential site over the other using in vitro mobility shift assays and nucleotide analog interference mapping of Xenopus U25 snoRNA and of a circularly permuted form. We find that preferential binding of human 15.5K is not dependent on the proximity of RNA ends, but instead appears to require a structural context beyond the kink-turn itself. Direct analysis of the energetic contributions to binding made by 18 functional groups within the kink-turn identified both backbone atoms and base functionalities as key for interaction. An intramolecular RNA-RNA contact via a 2'-hydroxyl may supercede a putative Type I A-minor interaction in stabilizing the RNA-protein complex.

Animals↗
PubMed2004

Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay.

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression.

Cell Line↗
PubMed2004

Guide RNAs with 5' caps and novel box C/D snoRNA-like domains for modification of snRNAs in metazoa.

BACKGROUND: Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important. The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles. Each particle is composed of a short guide RNA and a set of several proteins. All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns. RESULTS: We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs. Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA. These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure. The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures. A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron. CONCLUSIONS: This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed. Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.

Animals↗
PubMed2004

Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.

Immuno- and other affinity-purification approaches are commonly used to characterize the composition of ribonucleoprotein complexes. While associations detected by these procedures are often interpreted as reflecting in vivo interactions, it is also possible that they arise from reassociation of molecules after cell lysis. Here we used an experimental approach that allowed us to distinguish between these possibilities. Surprisingly, we show that the association of the RNA-binding protein HuR with its target mRNA, c-fos, as detected by co-immunoprecipitation, results largely from reassociation of molecules subsequent to cell lysis. The existence of such postlysis reassortments thus demonstrates that co-immunoprecipitation does not always recapitulate the in vivo state of ribonucleoprotein complexes.

Animals↗
PubMed2004

A molecular link between SR protein dephosphorylation and mRNA export.

In metazoans, multiple RNA-binding proteins, including the shuttling serine/arginine-rich (SR)-splicing factors, function as adapters for mRNA nuclear export by interacting with the export receptor TAP/nuclear export factor 1 (NXF1). Yet, it is unclear how interactions between adapters and TAP are regulated. Here, we demonstrate that the SR proteins 9G8 and ASF/SF2 exhibit higher affinity for TAP/NXF1 when hypophosphorylated. 9G8 is recruited to the pre-mRNA in a hyperphosphorylated form but becomes hypophosphorylated during splicing both in vivo and in vitro. TAP preferentially binds spliced mRNA-protein complexes compared with pre-mRNA-protein complexes. Thus, the phosphorylation state of the SR protein adapters may underlie the selectivity of TAP-mediated export of spliced mRNA.

Catalysis↗
PubMed2004

The Herpesvirus saimiri small nuclear RNAs recruit AU-rich element-binding proteins but do not alter host AU-rich element-containing mRNA levels in virally transformed T cells.

Herpesvirus saimiri (HVS) encodes seven Sm-class small nuclear RNAs, called HSURs (for Herpesvirus saimiri U RNAs), that are abundantly expressed in HVS-transformed, latently infected marmoset T cells but are of unknown function. HSURs 1, 2, and 5 have highly conserved 5'-end sequences containing the AUUUA pentamer characteristic of AU-rich elements (AREs) that regulate the stability of many host mRNAs, including those encoding most proto-oncogenes and cytokines. To test whether the ARE-containing HSURs act to sequester host proteins that regulate the decay of these mRNAs, we demonstrate their in vivo interaction with the ARE-binding proteins hnRNP D and HuR in HVS-transformed T cells using a new cross-linking assay. Comprehensive Northern and microarray analyses revealed, however, that the levels of endogenous ARE-containing mRNAs are not altered in T cells latently infected with HVS mutants lacking HSURs 1 and 2. HSUR 1 binds the destabilizing ARE-binding protein tristetraprolin induced following activation of HVS-transformed T cells, but even in such stimulated cells, the levels of host ARE-containing mRNAs are not altered by deletion of HSURs 1 and 2. Instead, HSUR 1 itself is degraded by an ARE-dependent pathway in HVS-transformed T cells, suggesting that HVS may take advantage of the host ARE-mediated mRNA decay pathway to regulate HSUR expression. This is the first example of posttranscriptional regulation of the expression of an Sm small nuclear RNA.

Animals↗
PubMed2004

An intronic enhancer regulates splicing of the twintron of Drosophila melanogaster prospero pre-mRNA by two different spliceosomes.

We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.

Animals↗