Maria M. Konarska

p54nrb associates with the 5′ splice site within large transcription/splicing complexes

The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54nrb, a transcription/splicing factor, associates with the 5′ splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO–snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P‐TEFb, TAT‐SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO–snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54nrb and PSF, which bind the C‐terminal domain of the largest subunit of RNAPII, can interact directly with the 5′ SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process.

“Nought May Endure but Mutability”: Spliceosome Dynamics and the Regulation of Splicing

The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing, or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction.

Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Communication between U1 and U2 snRNPs is critical during pre‐spliceosome assembly; yet, direct connections have not been observed. To investigate this assembly step, we focused on Prp5, an RNA‐dependent ATPase of the DExD/H family. We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function. Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre‐spliceosome complex A formation. hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1‐ and U2‐interacting domains that are required for pre‐spliceosome assembly; and, we observe a Prp5‐associated U1/U2 complex in S. pombe. Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre‐spliceosome formation.

Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.

PRPF8 defects cause missplicing in myeloid malignancies

Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Insights into Branch Nucleophile Positioning and Activation from an Orthogonal Pre-mRNA Splicing System in Yeast

The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second-copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic center. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research.

Splicing fidelity revisited

Since the discovery of Prp16, a spliceosomal ATPase that alters the fidelity of splicing, many other ATPases associated with the spliceosome have been postulated to work similarly. The finding that Prp22, a DEAH-box ATPase, functions as a fidelity factor for the second step of splicing supports this hypothesis.

A critical assessment of the utility of protein-free splicing systems

U2 and U6 snRNAs form part of the catalytic spliceosome and represent strong candidates for components of its active site. Over the past decade it has become clear that these snRNAs are capable of catalyzing several different chemical reactions, leading to the widespread conclusion that the spliceosome is a ribozyme. Here, we discuss the advances in both protein-free and fully spliceosomal systems that would be required to conclude that the reactions observed to be catalyzed by protein-free snRNAs are related to splicing and question the reliability of snRNA-only systems as tools for mechanistic splicing research.

Ligation of endogenous tRNA 3' half molecules to their corresponding 5' halves via 2'-phosphomonoester,3',5'-phosphodiester bonds in extracts of Chlamydomonas.

tRNA preparations from Chlamydomonas and wheat germ contain small amounts of tRNA 5′ halves and corresponding 3′ halves. Incubation of cell‐free extracts from the two sources with [γ‐32P]ATP yielded 5′‐32P‐labeled tRNA 3′ halves which were joined to their corresponding 5′ counterparts to form mature tRNA containing 2′‐phosphomonoester,3′, 5′‐phosphodiester bonds. tRNA 3′ halves labelled with T4 kinase were purified, sequenced and also joined to their 5′ counterparts. It is proposed that these tRNA halves may be intermediates of the tRNA splicing process, and that the RNA kinase and ligase activities observed here are part of the tRNA splicing complex.

Structural biology: Spliceosome's core exposed

The spliceosome complex removes intron sequences from RNA transcripts to form messenger RNA. The structure of a spliceosomal protein, Prp8, reveals the complexs active site and casts light on the origin of splicing. See Article p.638 The core of the spliceosome, a complex that removes introns from precursor mRNA transcripts prior to their expression, consists of several RNA–protein complexes arranged on the pre-mRNA. Prp8 is a protein in one such core complex, the U5 snRNP, and it contains the active site where cleavage occurs. Kiyoshi Nagai and colleagues have solved the structure of a large fragment of Prp8 bound to a U5 snRNP assembly factor, Aar2. This structure offers insight into how the splice sites might fit into the active site, and supports a possible unified evolutionary origin of eukaryotic pre-mRNA and bacterial group II intron-splicing mechanisms.