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Characterizing a cancer spliceosome Cells undergo many genomic changes as they progress toward metastatic cancer. One aspect of this change is to RNA expression and splicing isoforms, but how these differences affect tumor progression is not well characterized. Fish et al. developed a computational framework called pyTEISER that identifies structural cis-regulatory elements that control diverse types of RNA regulation. Applying pyTEISER to models of breast cancer metastasis, they discovered an RNA short-stem-loop element that forms a “structural splicing enhancer” that acts in cis to regulate alternative splicing of RNA transcripts. One of these interactions encompasses the RNA-binding protein SNRPA1 and results in alternative exon inclusion that affects metastatic capacity in xenograft models. Thus, RNA element binding may play a role in splicing regulation and is potentially an important component of the cis-splicing code. Science, this issue p. eabc7531 Studies of alternative splicing programs elucidate prometastatic mRNA isoforms in breast cancer. INTRODUCTION Alternative splicing is a posttranscriptional regulatory mechanism critical for transcriptome and proteome diversity. By increasing complexity at the protein level, alternative splicing can induce functional changes in the cell. It is well established that RNA structural elements play a critical role in posttranscriptional regulatory processes, including alternative splicing. Therefore, the role of regulatory information encoded by RNA secondary structure that governs alternative splicing decisions is of particular interest. Changes in alternative splicing patterns have been shown to govern cancer progression and metastasis, and therefore drivers of this process are of clinical interest. RATIONALE Pathological changes in alternative splicing patterns are considered a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. A major obstacle to better understanding these programs is that the bioinformatic strategies commonly used for the discovery of cis-regulatory elements fail to capture the contribution of RNA secondary structure to regulatory information. To address this, we had previously developed the computational framework TEISER (Tool for Eliciting Informative Structural Elements in RNA), which uses both RNA structural and sequence information to identify cis-regulatory elements that are informative of transcriptomic changes. Here, we introduce pyTEISER (pythonic TEISER), which incorporates experimentally derived and additional computationally predicted RNA structural information to investigate the RNA sequence and structural code that governs a broader range of RNA-related processes, including splicing and RNA processing, in addition to steady-state gene expression. RESULTS By applying pyTEISER to data from cell line and patient-derived xenograft models of breast cancer metastasis, as well as measurements in matched clinical samples from primary breast tumors and metastases, we have discovered and functionally characterized a previously unknown RNA structural element that acts as a splicing enhancer. We find that this structural element drives aberrant alternative splicing in highly metastatic breast cancer, and that the RNA-binding protein SNRPA1 (small nuclear ribonucleoprotein polypeptide A′) mediates this alternative splicing pathway through direct interactions with these structural elements, which we have named SNRPA1-associated structural splicing enhancers (S3Es). These elements are located near cassette exons that exhibit increased inclusion in cells with higher SNRPA1 expression. While SNRPA1 is a core component of the U2 snRNP, we provide evidence that SNRPA1 modulates the splicing of S3E-containing exons independently of its spliceosomal function. We identify the functional sequence and structure requirements for SNRPA1-S3E interactions in vitro and in vivo and demonstrate that this previously unknown SNRPA1-S3E regulatory pathway is up-regulated in highly metastatic breast cancer cells. We show that modulating SNRPA1 expression has a significant effect on the metastatic capacity of breast cancer cells by affecting their invasiveness. In contrast, SNRPA1 expression levels are not associated with changes in cell proliferation or primary tumor growth. Importantly, we identify PLEC as a target of SNRPA1-mediated alternative splicing and find that this SNRPA1-regulated alternatively spliced plectin isoform is also up-regulated in metastatic tumors. We show that correction of PLEC splicing using antisense morpholinos can reduce the metastatic capacity of breast cancer cells by down-regulating their invasiveness. CONCLUSION Our results establish a noncanonical function for SNRPA1 and a previously unknown RNA structural code that regulates alternative splicing, and we find that this SNRPA1-mediated pathway acts as a promoter of breast cancer metastasis. SNRPA1 uses an RNA structural code to drive a prometastatic alternative splicing program in breast cancer. Higher SNRPA1 expression in highly metastatic breast cancer cell lines and clinical samples increases the retention of a set of alternative cassette exons, driven by SNRPA1-associated structural splicing enhancer elements. PLEC is a functional target of SNRPA1, whose rod domain–containing isoform increases cancer cell invasion and promotes metastasis. Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A′ (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.