Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly

Kafasla, Panayiota; Patrinou-Georgoula, Meropi; Lewis, Joe; Guialis, Apostolia

doi:10.1042/bj3630793

Cited by 20 publications

(6 citation statements)

References 31 publications

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“…Because the hnRNP C proteins do not shuttle between the nucleus and the cytoplasm, they are apparently released from newly matured mRNAs within the nucleus or at the NPC. The hnRNP M proteins bind preferentially to poly(G)-and poly(U)-rich mRNA sequences (8,39), and like the C proteins they have also been implicated in regulating pre-mRNA splicing (20). It is currently unknown whether the hnRNP M proteins exit the nucleus with mRNPs or whether they are released from newly matured mRNAs in the nucleus or at the NPC.…”

Section: Discussionmentioning

confidence: 99%

SUMO Modification of Heterogeneous Nuclear Ribonucleoproteins

Vassileva

Matunis

2004

Molecular and Cellular Biology

102

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Small ubiquitin-related modifiers (SUMOs) are proteins that are posttranslationally conjugated to other cellular proteins, particularly those that localize and function in the nucleus. Enzymes regulating SUMO modification localize in part to nuclear pore complexes (NPCs), indicating that modification of some proteins may occur as they are translocated between the nucleus and the cytoplasm. Substrates that are regulated by SUMO modification at NPCs, however, have not been previously identified. Among the most abundant cargos transported through NPCs are the heterogeneous nuclear ribonucleoproteins (hnRNPs). HnRNPs are involved in various aspects of mRNA biogenesis, including regulation of pre-mRNA splicing and nuclear export. Here, we demonstrate that two subsets of hnRNPs, the hnRNP C and M proteins, are substrates for SUMO modification. We demonstrate that the hnRNP C proteins are modified by SUMO at a single lysine residue, K237, and that SUMO modification at this site decreases their binding to nucleic acids. We also show that Nup358, a SUMO E3 ligase associated with the cytoplasmic fibrils of NPCs, enhances the SUMO modification of the hnRNP C and M proteins. Based on our findings, we propose that SUMO modification of the hnRNP C and M proteins may occur at NPCs and facilitate the nucleocytoplasmic transport of mRNAs.The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of highly conserved RNA-binding proteins that have important roles in regulating multiple steps in mRNA biogenesis and function (10,11,23). In the nucleus, the hnRNPs form large complexes with primary RNA polymerase II transcripts that contain more than 20 different hnRNPs ranging in size from 30 to 120 kDa (4, 31). HnRNPs affect mRNA transcription (16, 28), regulate mRNA translation in the cytoplasm (12-14, 18, 21), and are involved in the maintenance of the single-stranded DNA (ssDNA) extensions at chromosome telomeres (7,(12)(13)(14)24). HnRNPs, however, are best known for their roles in regulating the nuclear posttranscriptional events involved in mRNA biogenesis, including regulation of pre-mRNA splicing, pre-mRNA polyadenylation, and 3Ј-end processing (1, 40) and mRNA nuclear export (10).With regard to their role in nuclear export, most hnRNPs remain associated with the mRNAs as they are translocated through nuclear pore complexes (NPCs) and into the cytoplasm (32, 41). Once in the cytoplasm, these hnRNPs are released from the mRNAs by an unknown mechanism and shuttle back into the nucleus. Intriguingly, several hnRNPs, including the hnRNP C proteins, do not shuttle between the nucleus and the cytoplasm and are presumably released from newly formed mRNAs in the nucleus prior to export (32). Exactly where and how the nonshuttling hnRNPs are released from mRNAs in the nucleus is not currently known. Although significant progress has been made in identifying the factors and steps involved in targeting nuclear mRNPs to NPCs for export, very little is still understood about the exact mechanisms involved in their translocat...

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Section: Discussionmentioning

confidence: 99%

SUMO Modification of Heterogeneous Nuclear Ribonucleoproteins

Vassileva

Matunis

2004

Molecular and Cellular Biology

102

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“…In our experiments, Drosophila ovarian, embryonic and S2-cell Rump protein migrate with an apparent mobility of ~70 kDa, consistent with its calculated molecular weight of 67 kDa. Mammalian hnRNP M proteins have been implicated in splicing through their association with pre-mRNA at an early step in spliceosome assembly (Kafasla et al, 2002). Studies in Chironomus have shown that Hrp59 binds to pre-mRNA co-transcriptionally and remains associated with the RNA until it reaches the nuclear envelope, and recent analysis of the Drosophila S2 cell protein shows that it regulates alternative splicing of its own mRNA (Hase et al, 2006;Kiesler et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

TheDrosophilahnRNP M homolog Rumpelstiltskin regulatesnanosmRNA localization

Jain

Gavis

2008

Development

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Anterior-posterior axis patterning of the Drosophila embryo requires Nanos activity selectively in the posterior. This spatial asymmetry of Nanos is generated by the localization of nanos mRNA to the posterior pole of the embryo, where it is subsequently translated. Posterior localization of nanos is mediated by a complex cis-acting localization signal in its 3Ј untranslated region comprising several partially redundant localization elements. This localization signal redundancy has hampered the identification of trans-acting factors that act specifically to effect posterior localization of nanos. Here, we have used a biochemical approach to identify Rumpelstiltskin, a Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) M homolog, which binds directly to an individual nanos localization element. Rumpelstiltskin associates with nanos mRNA in vitro and in vivo, and binding by Rumpelstiltskin correlates with localization element function in vivo. Through analysis of a rumpelstiltskin null mutation by genetic strategies that circumvent redundancy, we demonstrate that Rumpelstiltskin regulates anterior-posterior axis patterning by functioning as a direct-acting nanos mRNA localization factor.

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“…The hnRNP M protein contains an unusual repeat region rich in methionine and arginine residues that resembles a component of the cleavage stimulation factor (CstF) involved in polyadenylation (13). Since hnRNP M is transiently associated with the pre-mRNA at early stages of spliceosome assembly (27), it may thus act concurrently with several other WW domain-associated splicing factors. We observed that hnRNP M overexpression enhances exon 6 inclu- 5 HEK 293 cells.…”

Section: Discussionmentioning

confidence: 99%

The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

Lin

Tarn

2004

Molecular and Cellular Biology

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A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3 part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3 splice site recognition. Our results suggest an essential role of WW/FF domaincontaining factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Synthesis of mRNA by RNA polymerase II (Pol II) is coordinated with subsequent RNA processing events such as capping, splicing, and cleavage and polyadenylation (4,12,20,23,37). The largest subunit of RNA Pol II recruits mRNA processing activities during transcriptional elongation via its heptapeptide repeat-containing C-terminal domain (CTD) (4, 23). Truncation of the Pol II CTD results in reduced splicing in vivo (34). Under some circumstances, in vitro splicing of pre-mRNA can be stimulated by phosphorylated Pol II or CTD (22, 52). Several transcriptional activators or coactivators associate physically with small nuclear ribonucleoproteins (snRNPs) or serine/arginine-rich splicing factors (SR proteins) and thereby may modulate pre-mRNA splicing (18,29,35). Moreover, promoter structure affects alternative splice site selection during pre-mRNA splicing (11), and further analyses reveal that transcriptional activators differentially modulate the rate of transcriptional elongation, which in turn determines the outcome of two competing alternative splicing reactions (15,26,38). Nevertheless, several lines of evidence support the concept of a physical and functional coupling between transcription and splicing (12,20).Since the RNA Pol II CTD is implicated in promoting premRNA splicing, proteins that associate with the CTD may mediate this function. A two-hybrid screen previously revealed a set of RS domain-containing proteins via their interaction with the CTD (51). Amon...

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Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly

Cited by 20 publications

References 31 publications

SUMO Modification of Heterogeneous Nuclear Ribonucleoproteins

SUMO Modification of Heterogeneous Nuclear Ribonucleoproteins

TheDrosophilahnRNP M homolog Rumpelstiltskin regulatesnanosmRNA localization

The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

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