Spliceosomal U6 Small Nuclear RNA Research Articles

The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes

SummaryPat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Host RNA Packaging by Retroviruses: A Newly Synthesized Story.

ABSTRACTA fascinating aspect of retroviruses is their tendency to nonrandomly incorporate host cell RNAs into virions. In addition to the specific tRNAs that prime reverse transcription, all examined retroviruses selectively package multiple host cell noncoding RNAs (ncRNAs). Many of these ncRNAs appear to be encapsidated shortly after synthesis, before assembling with their normal protein partners. Remarkably, although some packaged ncRNAs, such as pre-tRNAs and the spliceosomal U6 small nuclear RNA (snRNA), were believed to reside exclusively within mammalian nuclei, it was demonstrated recently that the model retrovirus murine leukemia virus (MLV) packages these ncRNAs from a novel pathway in which unneeded nascent ncRNAs are exported to the cytoplasm for degradation. The finding that retroviruses package forms of ncRNAs that are rare in cells suggests several hypotheses for how these RNAs could assist retrovirus assembly and infectivity. Moreover, recent experiments in several laboratories have identified additional ways in which cellular ncRNAs may contribute to the retrovirus life cycle. This review focuses on the ncRNAs that are packaged by retroviruses and the ways in which both encapsidated ncRNAs and other cellular ncRNAs may contribute to retrovirus replication.

Molecular structures of unbound and transcribing RNA polymerase III.

Transcription of genes encoding small structured RNAs such as tRNAs, spliceosomal U6 snRNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. The cryo-EM structures of the S. cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, allow for the first time to build a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82/C34/C31 heterotrimer in close proximity to the stalk. The C53/C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.

It takes two to tangle: Prp24 and spliceosome assembly.

X-ray structural analysis of spliceosomal U6 small nuclear RNA bound to Prp24 reveals a complex interlocked RNA–protein assembly and suggests models for key steps in spliceosome assembly and recycling.

Site-specific labeling of RNA at internal ribose hydroxyl groups: terbium-assisted deoxyribozymes at work.

A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb(3+) as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2'-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2',5'-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ~160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.

Aberrant 3′ oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia

AbstractThe recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3′-5′ exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3′ end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2′, 3′ cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream. We show that the 3′ ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3′ oligo(A) tails that are characteristic of nuclear RNA surveillancetargets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3′-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN.

Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

Clericuzio-type poikiloderma with neutropenia (PN) is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA) posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3' end polyuridine tails that are longer than those in normal cells and lack a terminal 2',3' cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 isa 3'-to-5' RNA exonuclease that removes uridines from U6 3' ends, generating terminal 2',3' cyclic phosphates invitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3' end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

All small nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP localize to nucleoli; Identification of the nucleolar localization element of U6 snRNA.

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3' end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3'-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3' hydroxyl of U6 snRNA to a 3' phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies.

Cyclic 2′,3′-Phosphates and Nontemplated Nucleotides at the 3′ End of Spliceosomal U6 Small Nuclear RNA's

Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally. The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development. The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both).

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

A New U6 Small Nuclear Ribonucleoprotein-Specific Protein Conserved between cis- and trans-Splicing Systems

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA.

U6 small nuclear RNA (snRNA), a component of eukaryotic spliceosomes, is required for splicing of nuclear pre-mRNAs. Whereas trimethylguanosine cap-containing U sn-RNAs are transcribed by RNA polymerase II, the U6 RNA is transcribed by RNA polymerase III and contains a nonnucleotide cap structure on its 5' end. We characterized the cap structure of human U6 snRNA and show that the gamma phosphate of the 5' guanosine triphosphate is methylated. The mobilities of in vivo-modified gamma phosphate from the 5' end of HeLa U6 RNA were identical to the synthetic monomethyl phosphate (CH3-O-P) in two-dimensional chromatography and two-dimensional electrophoresis. The cap structure of U6 RNA is distinct from all other cap structures characterized thus far.