Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.

Pathogenic Abnormal Splicing Due to Intronic Deletions that Induce Biophysical Space Constraint for Spliceosome Assembly.

A precise genetic diagnosis is the single most important step for families with genetic disorders to enable personalized and preventative medicine. In addition to genetic variants in coding regions (exons) that can change a protein sequence, abnormal pre-mRNA splicing can be devastating for the encoded protein, inducing a frameshift or in-frame deletion/insertion of multiple residues. Non-coding variants that disrupt splicing are extremely challenging to identify. Stemming from an initial clinical discovery in two index Australian families, we define 25 families with genetic disorders caused by a class of pathogenic non-coding splice variant due to intronic deletions. These pathogenic intronic deletions spare all consensus splice motifs, though they critically shorten the minimal distance between the 5' splice-site (5'SS) and branchpoint. The mechanistic basis for abnormal splicing is due to biophysical constraint precluding U1/U2 spliceosome assembly, which stalls in A-complexes (that bridge the 5'SS and branchpoint). Substitution of deleted nucleotides with non-specific sequences restores spliceosome assembly and normal splicing, arguing against loss of an intronic element as the primary causal basis. Incremental lengthening of 5'SS-branchpoint length in our index EMD case subject defines 45-47 nt as the critical elongation enabling (inefficient) spliceosome assembly for EMD intron 5. The 5'SS-branchpoint space constraint mechanism, not currently factored by genomic informatics pipelines, is relevant to diagnosis and precision medicine across the breadth of Mendelian disorders and cancer genomics.

Smu1 and RED are required for activation of spliceosomal B complexes assembled on short introns.

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA's 5' splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5' and 3' regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5'SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.

The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation.

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion.

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport.

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.

Characterization of a short isoform of human Tgs1 hypermethylase associating with small nucleolar ribonucleoprotein core proteins and produced by limited proteolytic processing.

Tgs1 is the hypermethylase responsible for m(3)G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation.

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies.

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.

Characterization and expression analysis during embryo development of the mouse ortholog of MLL3.

We characterized the mouse ortholog of the human MLL3 gene and a 10.6 kb-Mll3 transcript. The mouse Mll3 gene comprises 60 exons that encompass 226 kb in chromosome 5. The predicted protein of 3464 amino acids contains two PHD domains, an ATPase alpha_beta signature, an HMG, and a SET domain. We analyzed the expression of the Mll3 gene during the embryonic development of the mouse by whole-mount in situ hybridization. Low levels of expression throughout the embryo were first detected at 8.0 dpc. At this stage, the signal was already stronger in the forebrain neuroepithelium and absent in the heart. Next, expression outlined the ventral neural tube, the somites, the limbs, and the eye lens remaining at low levels throughout the embryo. By 13.0 dpc, expression became stronger in the spinal cord, in hand/foot plates, and in gonads. RT-PCR confirmed that Mll3 is expressed early during gametogenesis. We suggest that Mll3 is expressed early in pre-spermatogonia and then in spermatogonia.

PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli.

To better understand intranuclear-targeting mechanisms, we have studied the transport of U3 snoRNA in human cells. Surprisingly, we found that PHAX, the snRNA export adaptor, is highly enriched in complexes containing m7G-capped U3 precursors. In contrast, the export receptor CRM1 is predominantly bound to TMG-capped U3 species. In agreement, PHAX does not export m7G-capped U3 precursors because their caps become hypermethylated in the nucleus. Inactivation of PHAX and CRM1 shows that U3 first requires PHAX to reach Cajal bodies, and then CRM1 to be routed from there to nucleoli. Furthermore, PHAX also binds the precursors of U8 and U13 box C/D snoRNAs and telomerase RNA. PHAX was previously shown to discriminate between small versus large RNAs during export. Our data indicate that the role of PHAX in determining the identity of small RNAs extends to nonexported species, and this appears critical to promote their transport within the nucleus.

Nuclear localization properties of a conserved protuberance in the Sm core complex.

The nuclear import signal of snRNPs is composed of two essential components, the m(3)G cap structure of the snRNA and the Sm core NLS carried by the Sm protein core complex. We have previously proposed that, in yeast, this last determinant is represented by a basic-rich protuberance formed by the C-terminal extensions of Sm proteins. In mammals, as well as in other organisms, this component has not yet been precisely defined. Using GFP-Sm fusion constructs and immunolocalization as well as biochemical experiments, we show here that the C-terminal domains of human SmD1 and SmD3 proteins possess nuclear localization properties. Deletions of these domains increase cytoplasmic fluorescence and cytoplasmic localization of GFP-Sm mutant fusion alleles. Our results are consistent with a model in which the Sm core NLS is evolutionarily conserved and composed of a basic-rich protuberance formed by C-terminal domains of different Sm subtypes.