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Why is there no U3 snRNP in the spliceosome complex of transcription initiation?

Why is there no U3 snRNP in the spliceosome complex of transcription initiation?


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snRNP U1,U2,U4,U5,U6 are present in the spliceosome but there is no mention of U3.


U3 RNA with its proteins forming a complex is localized at nucleolar. The box C'/D motif in U3 snRNA is responsible for this localization. Therefore U3 snRNA does not have a chance to go to mRNA transcription active sites. Instead, U3 snRNP play important roles in ribosomal-RNA processing. Because of necleolar localization, U3 snRNA is called U3 snoRNA alternatively.

https://en.wikipedia.org/wiki/Small_nucleolar_RNA_U3


Consider that all of the cells in a multicellular organism have arisen by division from a single fertilized egg and therefore, all have the same DNA. Division of that original fertilized egg produces, in the case of humans, over a trillion cells by the time a baby is produced from that egg (that's a lot of DNA replication!). Yet, we also know that a baby is not a giant ball of a trillion identical cells, but has many different kinds of cells that make up different tissues such as skin, muscle, bone, and nerves. How can cells that have identical DNA look so different and have different functions?

The answer lies in gene expression regulation, which is the process of using the information stored in DNA to generate different proteins through the steps of the central dogma of biology. Although all of the cells in a baby have the same DNA, each different cell type uses a unique subset of the genes in the DNA to direct the synthesis of a distinctive set of RNAs and proteins. The first step in gene expression is transcription.

What is transcription? Transcription is the process of copying information from DNA sequences into RNA sequences. This process is also known as DNA-dependent RNA synthesis. When a sequence of DNA is transcribed, only one portion of one of the two DNA strands is copied into RNA this is contrasted with the process of DNA replication (Chapter 14) where both strands of DNA must be copied, and in their entirety

Transcription copies short stretches of the coding regions (DNA sequence that is eventually made into proteins) of DNA to generate RNA. In humans, these short stretches can average about 8.5 kilobases (kb) and consist of both exons and introns. Exons contain the information to make proteins and introns are non-coding, intervening, DNA sequences. Different genes may be copied into RNA at varying times in the cell's life-cycle. RNAs are temporary copies of instructions of the information in DNA and different sets of instructions are copied for use at different times.

Table 3-1: General features of transcription.

The process of making RNA copies using a DNA as template is known as transcription

The process of transcription produces all sorts of RNAs (mRNA, tRNA, rRNA, etc)

One strand of the DNA serves as a template for the synthesis of RNA

Enzymes that synthesize RNA are called RNA polymerases

RNA polymerases synthesize RNA in the 5’ to 3’ direction

RNA polymerases do not need a primer

RNA polymerases uses rNTPs (ATP, GTP, UTP, and CTP) to build the new RNA strand

RNA polymerases bind at specific DNA sequences called promoters to start transcription

RNA polymerases stop RNA synthesis when they reach sequences called terminators

Cells make several different kinds of RNA:

Messenger RNA : mRNAs that code for proteins

Ribosomal RNA : rRNAs that form part of ribosomes

Transfer RNA : tRNAs that carry the amino acids to the ribosome during translation

MicroRNA s: miRNAs that regulate gene expression

Many other small RNAs that have a variety of unique functions, often, but not always, playing a role in regulating gene expression (ex. snRNA , snoRNA , TERC , lncRNA , piRNA , siRNA , and shRNA )

Table 3-2: Types of RNA polymerases found in eukaryotes and some of the RNAs they transcribe.

Building an RNA strand is very similar to building a DNA strand. This is not surprising, knowing that DNA and RNA are both nucleic acids, and as such are structurally very similar. Indeed, the three main differences between DNA and RNA are: 1) the presence of a 2’-OH group on the ribose sugar in RNA 2) uracil and not thymine is one of the pyrimidine nitrogenous bases in RNA and 3) DNA is double-stranded while the RNAs produced by transcription are single stranded. Transcription is catalyzed by the enzyme RNA Polymerase , which is a general term for an enzyme that makes a polymer of RNA. There are different RNA polymerases in eukaryotes that are responsible for synthesizing the many different RNAs found in cells (Table 3-2).

Figure 3-1: Bos taurus RNA polymerase II. The protein is shown in fuchsia DNA is the double helix structure shown in the protein. The backbones of the DNA are orange and light pink. The RNA being synthesized cannot be seen well at this angle but is coming out of the back of the protein structure.

Like DNA polymerases, RNA polymerases build new RNA molecules in the 5' to 3' direction, but because they are making RNA, they use ribonucleotides (i.e., RNA nucleotides) rather than deoxyribonucleotides (as in DNA). Ribonucleotides are joined in exactly the same way as deoxyribonucleotides, which is to say that the 3'-OH of the last nucleotide on the strand is joined to the 5' phosphate of the incoming nucleotide.

A critical difference between DNA polymerases and RNA polymerases is that the latter do not require a primer to start making RNA. Once RNA polymerases are in the right place to start copying DNA, they just begin synthesizing RNA by stringing together RNA nucleotides that are complementary to the DNA template. The fact that RNA polymerases can copy DNA without a primer is known as de novo RNA synthesis, meaning RNA synthesis “starts from the beginning”. In contrast DNA replication requires a primer to initiate DNA synthesis (Chapter 14).

Figure 3-2: RNA structure . RNAs have a hydroxyl group at the 2’-carbon, while DNA does not. RNAs use the nitrogenous base uracil, unlike DNA, which uses thymine. Additionally, the strand is generated in the 5’ to 3’ direction (in the direction of the red arrow), when the incoming nucleotide is added, its 5’ phosphate is linked to the 3’OH of nucleotide that is already incorporated into the RNA strand.


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Why is there no U3 snRNP in the spliceosome complex of transcription initiation? - Biology

To investigate the mechanism of spliceosome assembly in vivo, we performed chromatin immunoprecipitation (ChIP) analysis of U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) to intron-containing yeast (S. cerevisiae) genes. The snRNPs display patterns that indicate a cotranscriptional assembly model: U1 first, then U2, and the U4/U6•U5 tri-snRNP followed by U1 destabilization. cis-splicing mutations also support a role of U2 and/or the tri-snRNP in U1 destabilization. Moreover, they indicate that splicing efficiency has a major impact on cotranscriptional snRNP recruitment and suggest that cotranscriptional recruitment of U2 or the tri-snRNP is required to commit the pre-mRNA to splicing. Branchpoint (BP) mutations had a major effect on the U1 pattern, whereas 5′ splice site (5′ss) mutations had a stronger effect on the U2 pattern. A 5′ss-U1 snRNA complementation experiment suggests that pairing between U1 and the 5′ss occurs after U1 recruitment and contributes to a specific U1:substrate conformation required for efficient U2 and tri-snRNP recruitment.


Spliceosome Catalytic Cycle

The U1 and U2 snRNPs recognize the 5𠌪nd 3′ splice-sites (5′SS and 3′SS) and conserved branch sites (BS: AG) of introns. BS is followed by a polypyrimidine tract (PPT) upstream of the 3′SS in metazoans. BS and PPT are poorly conserved in plants (Brown and Simpson, 1998 Simpson et al., 2002), although orthologs of BS-interacting U2AF and PTB proteins are present in Arabidopsis. In vitro assembly studies indicate that the U1 snRNP binds first through U1-C and U1-70K to the 5′SS. Subsequently, PPT and 3′SS are bound respectively by the U2 auxiliary factors U2AF65 and U2AF35 that interact with SF1 at the BP, as well as with a range of SR, hnRNP, Transformer (Tra), and EJC proteins that recognize exonic and intronic splicing enhancer and silencer sequences, contributing to the definition of exons’ positions (for review see Will and Lührmann, 2011). The SR-related Arabidopsis SR45a/Tra-2b factor interacts, for example, with U1-70K and U2F35a to assist splice-site selection, as well as with PRP38 during spliceosome activation (Tanabe et al., 2009). From the 19 Arabidopsis SR-proteins classified into seven subfamilies (Barta et al., 2010), yeast and human orthologs of SR1, SC35, RS33, and RSZ33, as well as the SR-related proteins Tra-1A, B1/2, and SRm160 were identified in purified spliceosomal complexes (Table S1 in Supplementary Material). Transcription and alternative splicing of SR-protein genes is regulated by a multitude of stress and hormonal stimuli, and their known mutations result in pleiotropic regulatory defects (Reddy, 2007). The activity and stability of SR-proteins is regulated by phosphorylation including the Lammer/CLK, SRPK1, and SRPK2 kinases families, as well as by several PRMT arginine methylases that also recognize other classes (e.g., Sm, Lsm, hnRNP, etc.) of spliceosomal proteins (Fluhr, 2008). Arabidopsis PRMT5 was recently demonstrated to methylate several Sm and LSm factors (Deng et al., 2010). The prmt5 mutation results in defective splicing of FLK/hnRNP-E pre-mRNA and late flowering by increasing the FLC transcript level, as well as alters 5′SS recognition leading to aberrant processing of pre-mRNAs encoding components of the circadian clock (Sanchez et al., 2010).

Interaction of U1 with the U2AF-recruited U2 snRNP is stabilized by the ATP-dependent DExH/D-box RNA helicase Prp5. Displacement of SF1 by the SF3b14a subunit of PPT-binding U2-SF3a/b complex is stimulated by Prp5 leading to the formation of prespliceosome complex A (Behzadnia et al., 2007 Figure 2). Prp5 also facilitates annealing the U2 snRNA with BS, which bulges out an A residue of the intron for the first transesterification reaction. Subsequent recruitment of the U4/U6.U5 tri-snRNP results in the assembly of U1/2/4/5/6 penta-snRNP in the precatalytic complex B (Deckert et al., 2006). Penta-snRNP can be purified from yeast but it is inactive and requires additional factors, in particular the NTC, to form an activated B ACT complex (Stevens et al., 2002).

Figure 2. Schematic presentation of spliceosomal assembly and catalytic cycle. The scheme is drawn according to Wahl et al. (2009) indicating the assembly phase specific regulatory roles of key ATP-dependent DExH/D-box RNA helicases (in red) and the spliceosome-activating NTC complex. 5′ and 3′ splice site (5′SS and 3′SS), branch point (BP), and polypyrimidine tract (PPT). Exons are indicated by gray boxes, while thin black lines show intron and intron lariat.

During complex B to B ACT transition, interaction of U1 and U1-C with the 5′SS is interrupted by the Prp28/U5-100 helicase, which is activated by the U4/U6.U5 tri-snRNP-associated kinase SRPK2. Subsequently, unwinding the based-paired U4/U6 snRNAs by the Brr2/U5-200 helicase facilitates U6 snRNA interactions with the 5′SS and U2 snRNA. This stimulates the release of U1 and U4 snRNPs, U6 specific Lsm, and Prp24 proteins, as well as the formation of an intramolecular stem-loop (ISL) in U6, which represents the metal-binding catalytic center of the spliceosome. Brr2 is controlled by the interacting Snu114 GTPase and Prp8 U5 subunit. Removal of U2-associated SF3a/b proteins by the Prp2 helicase exposes BS in the remodeled catalytically active complex B*. During step I of splicing, the 2′-OH of BS adenosine residue attacks and forms a covalent bond with the U6 ISL-cleaved 5′SS yielding the 5′-exon and lariat intron-3𠌮xon intermediates.

Catalytic activation of the spliceosome critically depends on its association with the NTC before or during unwinding of the U4 and U6 snRNAs. The NTC regulates the interactions of U5 and U6 with the pre-mRNA before and after step I, as well as formation of the spliceosome’s catalytic center (Chan et al., 2003 Chan and Cheng, 2005). Nucleophilic attack of 3′-OH of 5′ exon at the 3′SS in step II requires further structural rearrangements by multiple factors leading to the formation of complex C. Prp8, anchoring the 5𠌮xon and lariat intron-3′ exon intermediates (Grainger and Beggs, 2005), the Prp16 ATPase, and the NTC subunit Isy1 are involved in monitoring completion of step I and displacing U6 from the 5′SS to liberate it for the catalytic step II. The Prp18 helicase and loop 1 of U5 snRNA juxtapose for ligation of the 5′ exon and 3′SS bound by the interacting NTC subunits Slu7 and Prp22 helicase (Smith et al., 2008). Next, Prp22 deposited downstream of the exon𠄾xon junction disrupts the interaction of Prp8 and U5 with exon sequences, releasing the spliced mRNA from complex C. Dissociation of U2, U5, and U6 is catalyzed by the Prp43 helicase, which is encoded by three candidates genes in Arabidopsis, while Brr2 and Snu114 are thought to unwind and separate the U2 and U6 components of the post-splicing complex (Valadkhan and Jaladat, 2010).


Gene Expression: Transcription of the Genetic Code

Termination

Termination of RNA transcription involves specific sequences downstream of the actual gene for the RNA to be transcribed. There are two types of termination mechanism—intrinsic termination and rho (ρ)—dependent termination. For the intrinsic termination, regions at the end of genes called terminator or termination sites can signal the termination of transcription. The termination sites are characterized by two inverted repeats spaced by a few other bases ( Fig. 3.10 ). The DNA then encodes a series of uracils. When the RNA is created, the inverted repeats form a hairpin loop. This tends to stall the advancement of RNA polymerase. Meanwhile, the presence of the uracils causes a series of A-U base pairs between the template strand and the RNA. Because A-U base pairs between the template strand and the RNA are weakly hydrogen-bonded compared with G-C pairs, the RNA dissociates from the transcription bubble and ends the transcription.

Fig. 3.10 . Intrinsic terminator sequence features—inverted repeats and a series of uracils.

The ρ-dependent termination involves an inverted repeat, so it also causes a hairpin loop to form but no string of U. In this case, the ρ protein binds to the RNA and chases the polymerase ( Fig. 3.11 ). When the polymerase transcribes the RNA that forms a hairpin loop, it stalls, giving the σ protein opportunity to catch up. When the σ protein reaches the termination site, it facilitates the dissociation of the transcription machinery. The result of the dissociation terminate the transcription.

Fig. 3.11 . Rho-dependent transcriptional termination.


RESULTS AND DISCUSSION

Identification of proteins that copurify with CBC

To identify protein interaction partners of CBC, a transgenic HeLa cell line stably expressing CBP20 with a C-terminal GFP-tag was established, using bacterial artificial chromosome (BAC) recombineering. BAC transgenesis offers the advantage that the “third allele” encoding the tagged protein of interest is expressed at physiological levels, due to usage of endogenous regulatory elements, and the GFP tag is effective for immunopurification and mass spectrometry (Poser et al. 2008). CBP20-GFP correctly localizes to the nucleus and binds both m 7 G caps and CBP80 (Supplemental Fig. 1A Pabis et al. 2010). Cell lysates were treated with benzonase to degrade DNA and RNA before immunopurification, thereby minimizing detection of proteins linked to CBC through nucleic acids (Cheeseman and Desai 2005 Pabis et al. 2010). Table 1 lists proteins specifically identified from both gel slice and shotgun preparations (see Supplemental Tables 1 and 2 for entire data sets). As expected, robustly detected proteins copurifying with CBP20 were CBP80, ARS2, PHAX, importins, and components of the recently characterized nuclear exosome targeting (NEXT) complex (Lubas et al. 2011).

TABLE 1.

Protein interactors of CBP20-GFP

The largest group of factors copurifying with CBP20 included 15 U1, U2, U5, U4/U6, and U4/U6·U5 snRNP-specific proteins. Identification of these proteins was highly specific, because many of the approximately 200 proteins present in the mammalian spliceosome (Wahl et al. 2009) were not detected moreover, highly abundant non-snRNP splicing factors (e.g., SR proteins) were absent. Despite the fact that the U4-, U5-, and U6-containing snRNPs are 10 times less abundant than U1 and U2 snRNPs (Yu et al. 1999), the number of peptides representing U4/U6·U5 snRNP components was remarkably high ( Table 1 ). Thus, the data suggest that CBC associates with all spliceosomal snRNPs, among them U4/U6·U5 snRNP in particular.

We focused on CBC interactions with snRNPs for validation by coimmunoprecipitation, using transgenic cell lines harboring GFP-tagged candidates (see Supplemental Fig. 1A𠄼 Sapra et al. 2009). It is possible that many proteins that coimmunoprecipitate with CBC are linked indirectly through either (pre-)mRNA or through the snRNAs contained in snRNPs. RNase digestion is able to remove both species of RNA and thereby abolish RNA-mediated interactions for example, we show that U1 snRNP is disrupted by RNase (Supplemental Fig. 1E Sapra et al. 2009). Therefore, cell lysates were treated with or without RNase A, and copurified material was probed for untagged CBP20 and CBP80 by Western blotting ( Fig. 1 , upper panel Supplemental Fig. 1D). As expected, CBP20 associations with CBP80 and ARS2 were RNase insensitive (Supplemental Fig. 1D). Similarly, CBC coimmunoprecipitated with the NEXT complex component ZC3H18/NHN1 from both mock and RNase-treated cell extracts ( Fig. 1 ). This observation, together with the report that NHN1 associates with mRNPs in a CBC-dependent manner (Merz et al. 2007), points to a potential role of CBC in recruiting NHN1 to the mRNP in vivo.

Validation of splicing factor binding to CBC. (Upper panel) Extracts from transgenic HeLa cells harboring GFP-tagged splicing factors, indicated left of the panel, were incubated with or without RNase A and subjected to immunoprecipitation with α-GFP. 0.6% of input (Inp) and 33% of the immunoprecipitate were analyzed by Western blot, using α-CBP20 and α-CBP80. Nonspecific background was assessed by immunoprecipitation with nonimmune IgG (IgG). See Supplemental Figure 1, D and E, for additional data and evidence that RNase treatment disrupted snRNPs. (Lower panel) Extracts from the same transgenic HeLa cells treated with and without α-amanitin to inhibit Pol II transcription, as indicated. All experiments were performed two to four times each, and representative gels are shown.

It was possible to validate the interactions of U1, U2, and U4/U6·U5 snRNPs with CBC, although many snRNP proteins appeared to be indirectly linked by RNA ( Table 1 ). Specifically, GFP-tagged U1 snRNP (U1-70K, U1A), U2 snRNP (SF3A1), and U4/U6·U5 snRNP (U5-200K, U4/U6-60K, U4/U6-61K, U5-116K, and Prp8) were all able to coimmunoprecipitate CBP20 and CBP80 in the absence of RNase ( Fig. 1 , upper panel Supplemental Fig. 1D,E). However, coimmunoprecipitation of U2AF65, U1-70K, U1-A, and SF3A1 was strongly reduced by RNase treatment, suggesting linkage through snRNA or pre-mRNA. In contrast, U5-200K (the human homolog of yeast Brr2, an RNA helicase), U4/U6-60K (hPrp4, a WD-repeat protein), and U4/U6-61K (hPrp31, a U4 snRNA-binding and Nop domain-containing protein) remained bound after RNase digestion, suggesting RNA-independent association of the U4/U6·U5 snRNP with CBC. To determine whether active transcription and splicing was strictly required for snRNP interactions with CBC, we treated cells with α-amanitin before cell lysis. Transcription inhibition did not disrupt interactions with these proteins. The insensitivity of U1-70K to α-amanitin ( Fig. 1 , lower panel) indicates that the loss of U1-70K upon RNase treatment ( Fig. 1 , upper panel) is indeed due to disruption of the U1 snRNP (Supplemental Fig. 1E). The continued detection of both CBP20 and CBP80 in immunoprecipitates of U1 and U4/U6·U5 snRNP proteins after transcription inhibition suggests that spliceosomal snRNPs may be constitutively associated with CBC. Alternatively, these interactions may be maintained by remaining unspliced RNA in the extract. The transcription- and RNA-independent interactions between CBC and snRNPs may explain why CBC is detected in purified spliceosomes assembled on pre-mRNAs that lack m 7 G caps (Fabrizio et al. 2009).

Taken together, this series of validation experiments indicates that CBC interacts in an RNA-independent manner with the U4/U6·U5 snRNP ( Table 1 ). There are no amino acid sequences or protein motifs shared among these partners, making it difficult to speculate on whether CBC association with these proteins is due to direct binding. Moreover, we cannot distinguish between binding to CBP20 and CBP80 in these experiments. In this study, we sought to understand the role of CBC in splicing, which requires spliceosome assembly from mature snRNPs. Therefore, we aimed to pursue the role of CBC in snRNP recruitment in vivo, rather than focus on CBC interactions with individual proteins. To do so, we coupled CBC depletion by RNAi with specific assays that interrogate the interactions of whole spliceosomal snRNPs with pre-mRNA.

SnRNP levels are unaffected by CBC depletion

Treatment of HeLa cells with CBP80-specific interfering RNAs by two independent methods was effective, leading to an �% reduction in CBP80 mRNA and protein levels ( Fig. 2 A Supplemental Fig. 2B,C). Importantly, CBP80 depletion also led to an �% reduction in CBP20 protein levels, resulting in an overall efficient knockdown of the whole CBC and indicating that CBP20 stability requires CBP80. This finding and the observed impairment of cell proliferation ( Fig. 2 A Supplemental Fig. 2A) are consistent with a prior study (Narita et al. 2007). Furthermore, expression levels of seven distinct splicing factors—including proteins copurifying with CBC, U1-70K, SF3A1, Prp8, and U5-116K—were unchanged upon CBC knockdown (Supplemental Fig. 2D), indicating that CBP80 depletion does not destabilize splicing factors in general.

Efficient CBC knockdown does not affect snRNP levels. HeLa cells transduced with retroviral vectors expressing CBP80 shRNA or without shRNA (Control) were assayed on day 6. (A) Assessment of CBC depletion through semiquantitative Western blotting (left panel) of CBP80, CBP20, and GAPDH after CBP80 knockdown (KD). Decreasing amounts of the same lysate were loaded. Comparison of changes in CBP80 and CBP20 RNA and protein levels (right panel). Band intensities were measured as integrated densities from Western blots and normalized to GAPDH. RNA levels were determined by RT-qPCR and normalized to 18S rRNA. N ≥ 8 different knockdowns. Error bars are the SEM. The changes are statistically significant (P < 0.005), as determined by the Student’s t-test. (B) Immunoprecipitation of snRNPs with α-TMG (K121), α-Sm proteins (Y12), and α-SART3. RNA was extracted, resolved on a 10% urea gel, and detected by Northern blot. A longer exposure of α-TMG IP lanes is shown.

Because these CBC depletion conditions were designed to test spliceosome assembly and snRNP dynamics in downstream assays, we were concerned that snRNP levels might be affected. CBC and PHAX binding to nuclear m 7 G-capped pre-snRNAs mediates their translocation to the cytoplasm, where Sm ring assembly and snRNA cap tri-methylation occur (Izaurralde et al. 1995 Ohno et al. 2000 Muller-McNicoll and Neugebauer 2013). If CBC knockdown were to impair snRNA export, reductions in cytoplasmic snRNP maturation steps and consequently cellular snRNP levels could occur. However, Northern blotting of total RNA revealed that levels of U1, U2, U4, and U6 snRNAs were similar among control and CBC-depleted cells ( Fig. 2 B). Importantly, levels of assembled snRNPs containing Sm proteins and tri-methyl guanosine (TMG) caps were unaffected. Quantitation revealed no significant differences in U1 and U2 levels between knockdown and control cells (Supplemental Fig. 3A). Because U4 and U6 snRNAs were less well detected by Northern blot, U4 and U6 snRNAs were immunoprecipitated with α-SART3, specific for the U4/U6 snRNP (Stanek et al. 2003). Detection of U4 and U6 snRNAs was robust and unchanged by CBC depletion ( Fig. 2 B Supplemental Fig. 3A). U5 snRNA levels were also not affected (Supplemental Fig. 3B). This surprising lack of effect on snRNP levels led us to verify CBC and PHAX binding to pre-snRNAs both CBC and PHAX pulled down pre-snRNAs, as expected (Supplemental Fig. 3C). Finally, metabolic labeling was performed to interrogate the degree of snRNA 5′-cap tri-methylation and showed no change upon CBC depletion (Supplemental Fig. 3D). We conclude that residual levels of CBC must be sufficient to support snRNA biogenesis in depleted cells. Because total spliceosomal snRNP levels were unaffected, it was possible to investigate a distinct role for CBC in splicing.

CBC depletion reduces cotranscriptional splicing

We selected the FOS gene to investigate the role of CBC in cotranscriptional splicing and spliceosome assembly for the following reasons. First, FOS transcripts are efficiently spliced cotranscriptionally (Listerman et al. 2006 Vargas et al. 2011). Second, FOS is strongly inducible (Piechaczyk and Blanchard 1994), permitting measurement of spliced and unspliced transcripts during the period of CBC depletion with minimal background from previously synthesized mRNA. Third, robust FOS transcription upon induction permits detection of splicing factor recruitment to the transcription unit by chromatin immunoprecipitation (ChIP) (Listerman et al. 2006 Sapra et al. 2009). Fourth, FOS is characterized by a relatively simple exon–intron structure ( Fig. 3 A). These features make FOS an ideal gene to study the effects of CBC depletion on splicing.

CBC depletion reduces cotranscriptional spliceosome assembly and splicing. HeLa cells transduced with control and α-CBP80 shRNA retroviral vectors were subjected to RNA isolation, ChRIP, or ChIP on day 6. (A) Schematic representation of FOS pre-mRNA and mRNA, showing the location of qPCR amplicons detecting RNA with either spliced or unspliced intron 1 (blue) and intron 3 (red). Total RNA (left panel) was reverse-transcribed with an oligo(dT) primer and the relative abundance of spliced to unspliced poly(A) + RNA assessed by qPCR. Nascent RNA (right panel) was isolated by ChRIP from an α-AcH4 IP and reverse-transcribed, using a primer in intron 2 (for intron 1 splicing) or in exon 4 (for intron 3 splicing). qPCR was conducted to assess the relative abundance of spliced to unspliced RNA. n ≥ 4 error bars are the SEM. (B) Schematic representation of the FOS gene. (Gray lines) The location of qPCR amplicons used in ChIP. The following antibodies were used: α-CBP80, α-Pol II-CTD (4H8), α-U2AF-65 (MC3), α-U5-116K, and α-GFP in case of transgenic cell lines expressing U1-70K-GFP or SF3A1-GFP. All values were calculated as percent input, normalized to nonimmune ChIP, and presented as fold enrichment over an intergenic region. In Pol II ChIP, normalization to intergenic was omitted due to absence of background. N ≥ 3 error bars are the SEM. Significant differences between knockdown and control determined by the Student’s t-test: (*) P < 0.05, (**) P < 0.01.

As a first step, we used ChIP to quantitate the impact of CBP80 RNAi on CBC levels at the FOS transcription unit. CBP80 ChIP confirmed that the �% decrease in cellular CBP80 protein corresponded to the reduction in CBP80 levels along the FOS gene ( Fig. 3 B). Acetylated histone H4 (AcH4) and Pol II levels were unchanged by CBC depletion ( Fig. 3 B Supplemental Fig. 4B), suggesting that FOS transcription is not CBC dependent. Next, we examined steady-state intron removal in FOS RNA by RT-qPCR, which revealed that the levels of unspliced FOS introns 1 and 3 increased by twofold upon CBC knockdown ( Fig. 3 A). In parallel, a decrease in total FOS mRNA was observed (Supplemental Fig. 4A). The levels of another short-lived, spliced mRNA, MYC, were also significantly reduced, and splicing of the first intron was inhibited by �% (data not shown). At the same time, mRNAs with long half-lives, such as ACTB, were unaffected by CBC depletion (Supplemental Fig. 4A), indicating that CBC depletion inhibits but does not abolish splicing activity. Another indication of cellular splicing activity is morphological: Nuclear speckles are normally interconnected, irregularly shaped nuclear compartments where splicing factors concentrate. Upon splicing inhibition with spliceostatin A, speckles become “rounded up” (Kaida et al. 2007). Supplemental Figure 5 shows that nuclear speckles also displayed a rounded-up morphology when cells were depleted of CBC, consistent with the expected overall splicing inhibition (see the Introduction). To determine the effect of CBC depletion on cotranscriptional splicing, nascent FOS RNA was retrieved by chromatin RNA immunoprecipitation (ChRIP), in which cross-linked active chromatin is immunoprecipitated with antibodies to AcH4 (Listerman et al. 2006). Figure 3 A shows that cotranscriptional splicing of FOS introns 1 and 3 was impaired upon CBC depletion, similar to total pre-mRNA splicing. This finding indicates that splicing inhibition is not likely due to effects on NEXT complex activity, because NEXT would not be expected to destabilize nascent RNA. We conclude that the absence of CBC leads to a decreased efficiency of cotranscriptional intron removal.

CBC depletion impairs cotranscriptional spliceosome assembly

Knowing that CBC is required for efficient nascent FOS RNA splicing, we sought to determine the step at which spliceosome assembly is altered in the absence of CBC. We therefore investigated the recruitment profiles of snRNPs, using a cotranscriptional spliceosome assembly assay based on ChIP (Gornemann et al. 2005 Listerman et al. 2006 Sapra et al. 2009). SnRNP-specific proteins were used as proxies for each snRNP: U1 (U1-70K), U2 (SF3A1), and U4/U6·U5 (U5-116K). GFP-tagged snRNP proteins expressed stably from BACs were incorporated into snRNPs and correctly localized (Supplemental Fig. 1A𠄼).

The levels and profiles of the U1, U2, and U4/U6·U5 snRNPs as well as U2AF65 were determined at four positions within exons and introns along the length of FOS ( Fig. 3 B). Upon CBC knockdown, accumulation of all three snRNPs was decreased ( Fig. 3 B). These effects are specific, because U2AF65 accumulation was unaffected. U5-116K accumulation was reduced significantly at the beginning as well as the end of FOS ( Fig. 3 B). The decrease in U4/U6·U5 snRNP recruitment is consistent with the notion that CBC associates with protein components of the tri-snRNP, namely, U5-200K, U4/U6-60K, and U4/U6-61K (see Fig. 1 Table 1 ) to assist spliceosome assembly. CBC depletion also caused marked reductions in U1 and U2 snRNP recruitment with accumulation of U1-70K-GFP 50% reduced at all positions ( Fig. 3 B). Because our results point to CBC interactions with U4/U6·U5 and not U1 snRNP proteins ( Fig. 1 Table 1 ), these data suggest that U4/U6·U5 snRNP association with CBC could assist U1 snRNP recruitment. Prior studies reported U5 snRNP interactions with the 5′ ss early in spliceosome assembly as well as U1 snRNP recruitment in the context of a multi-snRNP-containing complex (Wyatt et al. 1992 Malca et al. 2003). Thus, our data support the notion that CBC helps recruit U1 snRNP to the first 5′ ss (Lewis et al. 1996b), not by direct interactions with U1 but instead indirectly through the U4/U6·U5 snRNP. Moreover, the U1 snRNP is retained on multiple intron-containing pre-mRNAs after the first splicing event, rendering subsequent splicing more efficient (Crabb et al. 2010). Indeed, splicing of both FOS introns 1 and 3 was CBC dependent, suggesting—together with the observed snRNP profiles—that CBC may help retain the U1 snRNP on nascent RNA until all introns are removed. We conclude that proper cotranscriptional accumulation of U1, U2, and U4/U6·U5 snRNPs is dependent on CBC in vivo.

CBC promotes U1 and U5 snRNP interactions with pre-mRNA

The above data suggest that CBC’s overall role in FOS splicing is to enhance spliceosomal snRNP interactions with nascent RNA through its association with U4/U6·U5 snRNPs. To test this proposal more broadly, we used a live-cell imaging strategy to analyze the dynamic interaction of snRNPs with pre-mRNA both globally and at a different test gene. We addressed total cellular pre-mRNA by monitoring snRNP dynamics in the nucleoplasm complementary to this, we used an integrated, inducible transcription unit (E3) containing three exons and two introns of the β-globin gene ( Fig. 4 A Huranova et al. 2010 Pabis et al. 2010 Brody et al. 2011). E3 mRNA contains the MS2 binding site in the 3′ UTR, such that active transcription sites can be monitored in living cells with a fluorescently tagged MS2-binding protein. BACs harboring U1-70K-GFP, U2AF65-GFP, and Prp8-GFP were stably integrated into E3 cells, and fluorescence imaging revealed that U2AF65, U1, and U5 snRNPs were detectable in speckles and nucleoplasm, as expected ( Fig. 4 B). Importantly, each of these splicing factors accumulated at the E3 gene during active transcription, as seen by colocalization with E3 nascent RNAs at the transcription site ( Fig. 4 B).

CBC depletion reduces snRNP interactions with active transcription sites. (A) Diagram of the E3 construct stably integrated into U2OS cells. Expression is driven by a minimal CMV promoter (P) under control of the tetracycline response element and induced in the presence of doxycycline (dox) by the transactivator rtTA. The gene contains three β-globin exons (E1�) and a CFP coding sequence, 18 MS2 repeats, a polyadenylation cleavage signal, and a transcription terminator (T). (B) Imaging of nascent RNA at the E3 transcription unit in fixed cells by RNA FISH (Cy3-labeled probe to the MS2 sequence repeats red) together with the stably integrated U1 snRNP (U1-70K-GFP green), U2AF65-GFP, or U5 snRNP proteins (Prp8-GFP). Scale bars, 5 μm. (C) FRAP curves show recovery of U1-70K-GFP, U2AF65-GFP, and Prp8-GFP fluorescence at bleached spots placed in nucleoplasm, speckles, or at the E3 transcription site visualized by MS2BP-mCherry. Three independent experiments were performed in each 10� cells were tested. Error bars represent the SEM. For U1-70K-GFP and Prp8-GFP, the differences between recovery curves in control and CBC knockdown cells are statistically significant, as established using the Mann–Whitney test.

FRAP experiments revealed CBC-dependent dynamics among U1 and U4/U6·U5 snRNPs at both the E3 transcription site and in the nucleoplasm, where endogenous splicing occurs ( Fig. 4 C). In both locations, significantly faster recovery of both U1 and U5 snRNPs was observed upon CBC depletion, indicating that each snRNP was retained at sites of transcription for a shorter period of time. In contrast, mobility of U1 and U5 snRNPs in nuclear speckles was unchanged, suggesting that snRNP accumulation in speckles does not depend on interactions with CBC. No effects on U2AF65 recovery or mobility were observed at any location ( Fig. 4 C), in agreement with the ChIP data ( Fig. 3 B). Because the mobility of nucleoplasmic snRNPs is determined by association with pre-mRNA (Huranova et al. 2010), these data indicate that CBC knockdown weakens U1 and U4/U6·U5 snRNP interactions with endogenous pre-mRNAs in living cells. This finding is consistent with morphological evidence that splicing is globally inhibited (Supplemental Fig. 5).


Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions

During the final step in formation of a mature, functional mRNA, the introns are removed and exons are spliced together (see Figure 11-7). The discovery that introns are removed during splicing came from electron microscopy of RNA-DNA hybrids between adenovirus DNA and the mRNA encoding hexon, a major virion capsid protein (Figure 11-13). * Similar analyses of hybrids between RNA isolated from the nuclei of infected cells and viral DNA revealed RNAs that were colinear with the viral DNA (primary transcripts) and RNAs with one or two of the introns removed (processing intermediates). These results, together with the findings that the 59 cap and 39 poly(A) tail of mRNA precursors are retained in mature cytoplasmic mRNAs, led to the realization that introns are removed from primary transcripts as exons are spliced together. For short transcription units, RNA splicing usually follows cleavage and polyadenylation of the 3′ end of the primary transcript. But for long transcription units containing multiple exons, splicing of exons in the nascent RNA usually begins before transcription of the gene is complete.

Figure 11-13

Demonstration that introns are spliced out by electron microscopy of RNA-DNA hybrid between adenovirus DNA and the mRNA encoding hexon, a major viral protein. (a) Diagram of the EcoRI A fragment of adenovirus DNA, which extends from the left end of the (more. )

The location of exon-intron junctions (i.e., splice sites) in a pre-mRNA can be determined by comparing the sequence of genomic DNA with that of the cDNA prepared from the corresponding mRNA. Sequences that are present in the genomic DNA but absent from the cDNA represent introns and indicate the positions of splice sites. Such analysis of a large number of different mRNAs revealed moderately conserved, short consensus sequences at intron-exon boundaries in eukaryotic pre-mRNAs in higher organisms, a pyrimidine-rich region just upstream of the 3′ splice site also is common (Figure 11-14). The most conserved nucleotides are the (5′)GU and (3′)AG found at the ends of most introns. Deletion analyses of the center portion of introns in various pre-mRNAs have shown that generally only 30 –� nucleotides at each end of an intron are necessary for splicing to occur at normal rates.

Figure 11-14

Consensus sequences around 5′ and 3′ splice sites in vertebrate pre-mRNAs. The only nearly invariant bases are the (5′)GU and (3′)AG of the intron, although the flanking bases indicated are found at frequencies higher than (more. )

Recombinant DNAs containing the 5′ splice site of one transcription unit (e.g., SV40 late region) and the 3′ splice site of another (e.g., mouse β-globin gene) have been prepared and introduced into cultured cells. Spliced mRNA molecules are formed in which the two exon sequences are joined and the chimeric intron is deleted precisely. The formation of correctly spliced mRNAs in such experiments indicates that the cell’s splicing machinery can recognize 5′ and 3′ splice sites and correctly splice them together, with little influence from the intervening sequence in most cases.

Analysis of the intermediates formed during splicing of pre-mRNAs in vitro led to the conclusion that introns are removed as a lariat structure in which the 5′ G of the intron is joined in an unusual 2′,5′-phosphodiester bond to an adenosine near the 3′ end of the intron (Figure 11-15). This A residue is called the branch point because it forms an RNA branch in the lariat structure.

Figure 11-15

Analysis of RNA products formed in an in vitro splicing reaction. A nuclear extract from HeLa cells was incubated with a 497-nucleotide radiolabeled RNA (bottom) that contained portions of two exons (orange and tan) from human β-globin mRNA separated (more. )

The finding that excised introns have a branched lariat structure led to the discovery that splicing of exons proceeds via two sequential transesterification reactions (Figure 11-16). In each reaction, one phosphate-ester bond is exchanged for another. Since the number of phosphate-ester bonds in the molecule is not changed in either reaction, no energy is consumed. The net result of these two transesterification reactions is that two exons are ligated and the intervening intron is released as a branched lariat structure.

Figure 11-16

Splicing of exons in pre-mRNA occurs via two transesterification reactions. In the first reaction, the ester bond between the 5′ phosphorus of the intron and the 3′ oxygen (red) of exon 1 is exchanged for an ester bond with the 2′ (more. )


INTRODUCTION

Pre-mRNA splicing is a two-step transesterification reaction catalyzed by the spliceosome, a large complex assembled from preformed subcomplexes, called spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and hundreds of additional proteins (Jurica and Moore, 2003). In turn, the five major spliceosomal snRNPs, U1, U2, U4, U5, and U6, each consist of a single small nuclear RNA (snRNA) and specific set of proteins. Among the shared protein components of snRNPs are the seven Sm proteins, which are assembled as a stable, heteroheptameric ring on the RNA polymerase II-transcribed snRNAs: U1, U2, U4, and U5. After transcription, these snRNAs are transported to the cytoplasm, where the Sm ring is assembled on snRNAs by the SMN complex. Subsequently, the 5′ ends of the snRNAs are hypermethylated to generate the trimethyl-guanosine cap, which together with SMN, promotes snRNP nuclear import (reviewed in Will and Luhrmann, 2001 Matera and Shpargel, 2006 Tycowski et al., 2006). Because snRNPs do not shuttle between the nucleus and cytoplasm (Änkö and Neugebauer, unpublished data), Sm ring assembly seems to occur early and only once in the lifetime of each snRNP. A related heteroheptameric ring, consisting of seven “like-Sm” (LSm) proteins, is assembled on U6, an RNA polymerase III transcript, which is thought to remain in the nucleus for all assembly steps (Achsel et al., 1999 Mayes et al., 1999 Kiss, 2004 Listerman et al., 2007).

Once back in the cell nucleus, snRNPs first accumulate in CBs before distributing throughout the nucleoplasm, where splicing occurs (Sleeman and Lamond, 1999 Sleeman et al., 2001 Neugebauer, 2002). This suggests a role for CBs in nuclear steps of snRNP maturation, a prediction borne out by the following set of observations. First, posttranscriptional modifications of the snRNAs themselves occur in CBs after snRNP reimport from the cytoplasm (Darzacq et al., 2002 Kiss, 2002 Jady et al., 2003). These modifications, including pseudouridinylation and 2′-O-methylation, are guided by small Cajal body-specific RNAs. Second, CBs are the site of complex assembly steps that involve RNA–RNA annealing and the sequential addition of proteins. For example, the U4/U6 snRNP is formed when the U4 and U6 snRNAs anneal, a step catalyzed by U6-specific LSm proteins and SART3 (also named hPrp24 or p110) (Ghetti et al., 1995 Raghunathan and Guthrie, 1998 Achsel et al., 1999 Bell et al., 2002). Subsequently, the U4/U6·U5 tri-snRNP assembles when U5 snRNP associates by protein–protein interactions with the U4/U6 snRNP (Makarova et al., 2002 Schaffert et al., 2004). Both U4/U6 and U4/U6·U5 tri-snRNP assembly occur in CBs (Schaffert et al., 2004 Stanek and Neugebauer, 2004). Recently, mathematical modeling of U4/U6 snRNP formation in the cell nucleus revealed that accumulation of U4 and U6 snRNPs in CBs should greatly increase the efficiency of U4/U6 assembly (Klingauf et al., 2006). An additional role of CBs in U2 snRNP formation (Nesic et al., 2004) further points to CBs as the key site of nuclear steps in snRNP assembly. The observation that depletion of coilin, a protein required for snRNP concentration in CBs, impairs cell proliferation (Lemm et al., 2006) is consistent with the proposal that snRNP assembly is inefficient in the absence of CBs.

snRNPs must not only assemble de novo but also may regenerate after splicing to complete the so-called spliceosome cycle. During spliceosome assembly and activation, snRNPs undergo structural rearrangements, including U4/U6 snRNA unwinding and release of the U4 snRNP from the spliceosome (Staley and Guthrie, 1998). After splicing, mRNA is released from the spliceosome by the DEAH-box helicase hPrp22/HRH1 and snRNPs remain associated with the excised intron lariat (Company et al., 1991 Ohno and Shimura, 1996). In Saccharomyces cerevisiae, a complex of three proteins (Prp43/Ntr1/Ntr2) was shown to be essential for release of individual snRNPs from the lariat (Arenas and Abelson, 1997 Martin et al., 2002 Tsai et al., 2005 Boon et al., 2006 Tanaka et al., 2007 Tsai et al., 2007). If these released snRNPs are to participate in subsequent rounds of splicing, they have to be reassembled into the active U4/U6·U5 tri-snRNP. Several studies provide genetic and biochemical evidence for snRNP reassembly (Raghunathan and Guthrie, 1998 Bell et al., 2002 Verdone et al., 2004 Chen et al., 2006). Although snRNPs are highly expressed, the long half-lives of snRNAs suggests that they likely recycle and function again (Yu et al., 1999).

In the present study, we address the hypothesis that snRNPs cycle more than once through CBs. We show in living cells that CBs contain mostly mature snRNPs, which are capable of exchanging with nucleoplasm and visiting multiple CBs. Targeted knockdown of proteins involved in spliceosome recycling, hPrp22, and the human homologue of the recently identified yeast Ntr1, led to a dramatic accumulation of the U4/U6 snRNP in CBs. These data demonstrate that the CB is a vital way station in the spliceosomal cycle.


Materials and methods

Yeast strain construction

Gene disruption and tagging on the chromosome were performed using PCR fragments following a published strategy ( Puig et al, 1998 ) SNU17, BUD13 and PML1 genes were disrupted with the S. pombe HIS3 marker from pFA-HIS3MX6 ( Wach et al, 1997 ) in the haploid strain BMA64-1a (MATa, ura3-1, trp1- 2, leu2-3,112, his3-11, ade2-1, can1-101). RDS3 and YSF3 genes were disrupted by integrating K. tactis TRP1 marker from pBS1479 in the diploid strain BSY320 (ade2, arg4, leu2-3 112, trp1-289, ura3-52).

Ysf3 TAP, Snu17 TAP, Bud13 TAP and Pml1 TAP strains were constructed as described previously ( Rigaut et al, 1999 ). The TAP Rse1 fusion strain was described previously ( Puig et al, 2001 ).The Δmlp1 strain was described ( Galy et al, 2004 ).

TAP purification

All TAP purifications were performed as described previously from 2 l of culture ( Rigaut et al, 1999 ). Purified proteins were concentrated by lyophilization, separated by SDS–PAGE and stained with Coomassie Blue or silver.

Mass spectrometry analysis

Proteins were identified following ‘in gel’ digestion ( Pandey and Mann, 2000 Godovac-Zimmermann and Brown, 2001 Rappsilber and Mann, 2002 ). Coomassie-stained gels were treated directly, while silver-stained bands were rapidly destained using the Silver Quest decoloration kit (Sigma Aldrich). Digestion was carried out overnight with the addition of 50 ng of trypsin. A MALDI-Tof mass spectrometer (Voyager DE STR, Applied Biosystem) fitted with a pulsed nitrogen laser (337 nm) was used. Mass spectra were acquired in the reflectron mode. The total acceleration voltage was 20 kV, with a grid voltage of 68% and a delay extraction of 240 ns. Close external calibration was realized using a standard peptide mix solution ranging from 573 to 3496 Da (LaserBio Labs, SophiaAntipolis). Samples were prepared in the CHCA matrix at a final concentration of 10 mg/ml in acetonitrile/trifluoroacetic acid (70/0.1%) solution. In all, 1 μl of this sample solution corresponding to a dilution of 1:2 was then deposited on the MALDI target and dried.

Database scans were performed by using MS Fit and Profound search engines. Protein identifications were obtained with a sequence coverage of 55–86% in average, and mass accuracies of about 15–60 ppm.

In vitro splicing analysis

Splicing reactions were as described before, except that the incubation was performed for 30 min at 25°C ( Séraphin et al, 1988 ). Pre-mRNA was generated by in vitro transcription of plasmid pBS195 (wild type) or pBS199 (ΔUACUAAC) digested with DdeI. Reactions were stopped by addition of 200 μl of PK buffer (0.1 M Tris–HCl (pH 7.5), 12.5 mM EDTA (pH 8.0), 150 mM NaCl, 1% SDS) containing 80 μg of proteinase K (Sigma) and 10 μg of Escherichia coli tRNA. After incubation for 20 min at 37°C, RNA was extracted and analysed in a 15% polyacrylamide–7 M urea gel.

For complementation of in vitro splicing reaction with purified RES complex, it was TAP purified and dialysed against buffer D (20 mM HEPES-KOH (pH 7.9), 150 mM KCl, 8% glycerol, 0.5 mM phenylmethylsulphonyl fluoride and 0.5 mM dithiothreitol. A volume of 1 μl of purified material (or as control buffer D) was added to 10 μl splicing reactions.

Immunoprecipitation and primer extension

Immunoprecipitation and primer extension were as described previously ( Séraphin, 1995 ). Immunoprecipitation of pre-mRNA was performed similarly: Briefly, 50 μl splicing reactions were preformed. In all, 10 μl of the reaction was extracted and kept as an input, while the remaining 40 μl was diluted in 500 μl of IPP150 buffer (10 mM Tris (pH 8), 150 mM NaCl, 0.1% NP40) and used for immunoprecipitation.

In vivo splicing assays

The Δsnu17, Δbud13, Δpml1 strains and an isogenic control were transformed with reporters: RP51A wild-type intron (HZ18), 5′II (HZ12) ( Jacquier et al, 1985 ), pre-mRNA in-frame (pLG-Nde°Acc°), mRNA in-frame or no intron (pLG-SD5) ( Jacquier et al, 1985 Rain and Legrain, 1997 ). Reporters were assayed for β-galactosidase activity at two temperatures, 25 and 37°C. Strains were grown overnight at 25°C in a synthetic medium without uracil containing 2% lactate (pH 5.5), 2% glycerol and 0.05% glucose to an OD600 of 0.5–0.8. Cultures were maintained at 25°C or shifted to 37°C for 1 h before a 2 h induction of β-galactosidase. β-Galactosidase activity was tested as described previously ( Rutz and Séraphin, 2000 ). All the experiments were performed in duplicate using two independent transformants. Error bars present standard deviation. Primer extensions to analyse splicing and retention reporters were performed as described previously ( Jacquier et al, 1985 ).

Biocomputing

BLAST was used for database searches. Multiple sequence alignments were done with ClustalW. The neighbour-joining tree was computed, excluding positions with gaps and correcting for multiple substitutions using Clustal W ( Thompson et al, 1994 ).



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